Cyclin E genes and proteins

ABSTRACT

Disclosed are nucleic acid molecules encoding novel cyclin E2 polypeptides. Also disclosed are methods of preparing the nucleic acid molecules and polypeptides, and methods of using these molecules.

This application is a division of application Ser. No. 09/092,770 filedJun. 5, 1998, which is hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to novel genes encoding proteins thatare members of the cell cycle protein family known as "cyclins". Morespecifically, the invention is directed to a novel protein called cyclinE2, DNA encoding cyclin E2, and methods of making and using the cyclinE2 genes and polypeptides.

BACKGROUND Related Art

A. CDK-Cyclin Complexes

Cell division is a complex process that is regulated by a number ofcellular and environmental factors. Recent studies have identified twoclasses of proteins that appear to play key roles in controlling thecell cycle. These classes include the cyclin-dependent protein kinases("cdks") and the cyclins. Cdks function by phosphorylating selectedprotein substrates in the cell; these phosphorylated proteins in turn"signal" the cell to either enter or continue the process of celldivision. For cdks to be active, i.e., to phosphorylate other proteins,they must be bound to a cyclin protein. Thus, cyclins "regulate" theactivity of cdks by binding to them.

Several cdks and cyclins have been identified in mammals. At present, 9cdks are known, and they are referred to as "cdk1", "cdk2", and so on.Ten families of cyclins are currently recognized, and are referred to as"cyclin A", "cyclin B", and so forth through "cyclin J". For a generalreview of cyclins see Coats et al. (in Signal Transduction, Heldin andPurton, eds., Chapman and Hall, publishers {1996}; pages 347-360) andLees (Curr. Opinions Cell Biol., 7:773-780 [1995]).

Each cyclin family may have more than one member. Mammals, for example,have two types of cyclin A; cyclin Al and cyclin A2, and three types ofcyclin D (D1, D2, and D3). Prior to the present invention, only onemammalian cyclin E, cyclin E1, was known, although Zariwala et al.(Pathways to Cancer, a Cold Spring Harbor Winter Conference, Harlow etal., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.p. 49 [1998]) have purportedly identified a novel cyclin E protein. NoDNA or amino acid sequence data regarding this molecule is available.

Cyclin family members are typically classified based on their amino acidsequence homology to existing family members. For example, cyclin A2 hasabout 45 percent amino acid sequence homology to cyclin A1, but onlyabout 19 percent sequence homology to cyclin D1 and 21 percent sequencehomology to cyclin E1 (as calculated using the MacVector® clustalalignment software program from Oxford Molecular Group).

All cyclin molecules contain an amino acid sequence domain referred toas the "cyclin box". The cyclin box is about 100 amino acids in length(the average full length of a cyclin polypeptide is 300-500 amino acids)and is located in the middle portion of each cyclin. While the preciseamino acid sequence of the cyclin box varies from family to family, andeven within members of a family, there is a highly conserved motifwithin the cyclin box that is consistently present in all cyclin boxes(see Prosite public database, accession number PS00292 which sets fortha cyclin box "consensus" sequence).

Cyclins and cdks bind to each other in a highly selective manner; notall cyclins bind to all cdks. For example, cyclin D1 can associate withcdk4, but not with cdk2. Similarly, cyclin E can associate with cdk2 andcdk3, but not with cdk4; cyclin A can associate with cdk2, but not withcdk5. Formation of cyclin-cdk complexes is transient; the two moleculesmay be present in the cell at the same time, but can only form an activecomplex if cdk is phosphorylated by an enzyme referred to as "cak" forcdk activating kinase.

B. Cyclin E1

Human cyclin E1 was first cloned in 1991 and was found to bind to andactivate cdk2 (U.S. Pat. No. 5,449,755 issued Sep. 12, 1995; WO 93/06123published Apr. 1, 1993; Koff et al., Cell, 66:1217-1228 [1991]; see alsoPCT patent application WO 98/03649, published Jan. 29, 1998). Cyclin E1homologs have been identified in Drosophila (Richardson et al.,Development, 119:673-690 [1993]), mouse (Damjanov et al., Biochem.Biophys. Res. Comm., 201:994-1000 [1994), Xenopus (Chevalier et al., J.Cell Sci., 109:1173-1184 [1996]) and Zebrafish (Yarden et al., Devel.Dynam. 206:1-11 [1996]). In addition, two cyclin E1 variants have beenreported. The first of these is a human cyclin E1 splice variant(Mumberg et al., Nuc. Acids Res., 25:2098-2105 1997]) that purportedlyhas an internal deletion of 45 amino acids, and has an expressionpattern that is distinct from full length cyclin E1. The other reportedvariant purportedly lacks the 15 amino acids at the amino terminus(Ohtsubo et al., Mol. Cell. Biol., 15:2612-2624 [1995]).

A novel cyclin polypeptide, called cyclin N, that purportedly is relatedto cyclin E1 has recently been reported (Lauper et al., Abstracts fromthe 1998 Cold Spring Harbor Laboratory Cell Cycle Meeting, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. [1998] p. 115).

C. Cyclins and Cancer

One hallmark of cancer is the uncontrolled, and seemingly unregulated,division of cells. The role of cyclins and cdks in regulation of celldivision suggests that these proteins may be involved in convertingnormal cells to cancerous cells. Numerous recent studies have thusfocused on cyclin and cdk involvement in cancer. For example, in arecent review article, Sherr (Science, 274:1672-1677 [1996]) pointed outthat overexpression of cyclin D1 is seen in sarcomas, colorectal tumors,and melanomas, and that cyclin E is overexpressed in breast, stomach,colon and endometrial carcinomas.

Sarcevic et al. (J. Biol. Chem., 272:33327-33337 [1997]) describe thesubstrate specificies of various cyclin-cdk complexes in T-47D humanbreast cancer cells. They found, for example, that cyclin D1-cdk4phosphorylated a 38 kDa protein, while cyclin D3-cdk4 phosphorylated a105 kDa protein, a 102 kDa protein, and a 42 kDa protein. Cyclin E1-cdk2and cyclin A-cdk2 phosphorylated several proteins.

Cyclin E1 has been implicated in breast cancer. In young breast cancerpatients, high cyclin E1 expression in breast tumor tissue purportedlycorrelates with decreased survival (Porter et al., Nature Med.,3:222-225 [1997]). In addition, cyclin E1 is apparently overexpressed invarious breast cancer cell lines (Gray-Bablin et al., Proc. Natl. Acad.Sci USA, 93:15215-15220 [1996]).

Given that cyclin-cdk complexes are involved in cell division and areactive in cancer cells, inactivation of these complexes could result indecreased tumor cell proliferation. U.S. Pat. No. 5,645,999 issued Jul.8, 1997 describes assays that are purportedly useful for identifyingcompounds that modulate cyclin E1 activity.

A few naturally occurring proteins have been identified as inhibitors ofcyclin-cdk activity. These include the proteins p21 and p27 (U.S. Pat.No. 5,688,665 issued Nov. 18, 1997; PCT W096/02140, published Feb. 1,1996; Sherr et al., Genes and Devel., 9:1149-1163 [1995]). The proteinp21 purportedly binds directly to cdk1, cdk2, and cdk4, and canapparently inhibit both cyclin D-cdk complexes and cyclin E-cdkcomplexes (Sherr et al., supra). The protein p27 purportedly binds tocyclin-cdk complexes (rather than to isolated cdks) and can apparentlyinhibit cyclin A, B, D and E dependent kinase activity (Sherr et al.,supra). However, cyclin E1-cdk2 has also been found to purportedlyregulate p27 (Sheaff et al., Genes and Devel., 11:1464-1478 [1997]).

In view of the devastating effects of cancer, there is a need in the artto identify molecules in the human body which may have an important rolein the etiology of this disease, and to manipulate the expression ofsuch molecules in patients suffering from these and related diseases.

Accordingly, it is an object of this invention to provide nucleic acidmolecules and polypeptides that have a role in cell division.

It is a further object to provide methods of altering the level ofexpression and/or activity of such polypeptides in the human body.

Other related objects will readily be apparent from a reading of thisdisclosure.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides an isolatedbiologically active cyclin E2 nucleic acid molecule encoding apolypeptide selected from the group consisting of the nucleic acidmolecule comprising SEQ ID NO:1; the nucleic acid molecule comprisingSEQ ID NO:2; the nucleic acid molecule comprising SEQ ID NO:5; thenucleic acid molecule comprising SEQ ID NO:7; the nucleic acid moleculecomprising SEQ ID NO:8; a nucleic acid molecule encoding the polypeptideof SEQ ID NO:3, or a biologically active fragment thereof; a nucleicacid molecule encoding the polypeptide of SEQ ID NO:4, or a biologicallyactive fragment thereof; a nucleic acid molecule encoding thepolypeptide of SEQ ID NO:6, or a biologically active fragment thereof; anucleic acid molecule that encodes a polypeptide that is at least 70percent identical to the polypeptide of SEQ ID NO:1; a nucleic acidmolecule that encodes a polypeptide that is at least 70 percentidentical to the polypeptide of SEQ ID NO:5; a nucleic acid moleculethat hybridizes under conditions of high stringency to any of thenucleic acids above; and an isolated nucleic acid molecule that is thecomplement of the nucleic acid molecules above.

In another embodiment, the present invention provides vectors comprisingthe nucleic acids, and host cells comprising the vectors.

In yet another embodiment, the invention provides a process forproducing a cyclin E1 polypeptide comprising the steps of expressing apolypeptide encoded by cyclin E2 nucleic acid molecule in a suitablehost cell and isolating the polypeptide.

In yet another embodiment, the invention provides a cyclin E2polypeptide selected from the group consisting of: the polypeptide ofSEQ ID NO:3, the polypeptide of SEQ ID NO:4, the polypeptide of SEQ IDNO:6, and a polypeptide that is at least 70 percent identical to any ofSEQ ID NO:3, 4, or 5, wherein the cyclin E2 polypeptide may or may nothave an amino terminal methionine.

In one other embodiment, the present invention provides a method ofincreasing proliferation of a cell, comprising expressing a nucleic acidencoding cyclin E2 or a biologically active fragment thereof, in thecell.

In still a further embodiment, the present invention provides a methodof increasing cell division of a cell, comprising expressing a cyclin E2gene, or a biologically active fragment thereof, in the cell.

The invention further proves a method of decreasing cell division in acell, comprising expressing a cyclin E2 mutant in a cell, wherein themutant does not have cyclin E2 biological activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the full length cDNA sequence of human cyclin E2 (SEQ IDNO:1).

FIG. 2 depicts the full length cDNA sequence of mouse cyclin E2 (SEQ IDNO:2).

FIG. 3 depicts the putative full length amino acid sequence (SEQ IDNO:3) of human cyclin E2 as translated from the cDNA sequence.

FIG. 4 depicts the putative full length amino acid sequence (SEQ IDNO:4) of mouse cyclin E2 as translated from the cDNA sequence.

FIG. 5 depicts the full length cDNA sequence of a human cyclin E2 splicevariant (SEQ ID NO:5).

FIG. 6 depicts the putative full length amino acid sequence (SEQ IDNO:6) of the human cyclin E2 splice variant encoded by the DNA of SEQ IDNO:5.

FIG. 7 depicts the sequence of a DNA molecule (SEQ ID NO:7) encodingfull length human cyclin E2 in which the codons have been optimized forexpression in E. coli cells.

FIG. 8 depicts the sequence of a DNA molecule (SEQ ID NO:18) encodingfull length mouse cyclin E2 in which the codons have been optimized forexpression in E. coli cells.

FIG. 9 is a photograph of a Coomassie stained SDS gel. "Stds" refers toprestained molecular weight standards of the indicated molecularweights, and "Cyc E2" refers to human cyclin E2 polypeptide.

FIGS. 10A-10E are photographs of Western blots (10A-D) and an autorad(10E). Human cells were transfected with various DNA constructs asindicated at the bottom of the panels. "pEGFP" refers to the GFP vector;"HA-cdk2" refers to hemagluttinin-tagged cdk2; "p27" refers to a vectorcontaining human p27 cDNA; "GFP-E2" refers to a vector containing GFPDNA fused to full length cyclin E2 cDNA; and "GFP-E2sv" refers to avector containing GFP DNA fused to human cyclin E2 splice variant cDNA.For all panels, the transfected cells were lysed. In 10A-D, the celllysates were treated with anti-GFP antiserum after which ProteinA-Sepharose beads were added to immunoprecipitate the anti-GFP antibody.Immunoprecipitate was run on a gel, transferred to a Western blot, andprobed with the antibody indicated to the left of each panel. For 10E,histone H1 and 32P-ATP were added to the cell extract, incubated forabout 30 minutes at room temperature, and the mixture was then run onSDS-PAGE. The gel was then exposed to film for 2 hours. The identity ofeach band on the Western blots and autorad is indicated on the right ofeach panel.

FIG. 11 depicts an "ambiguous sequence" of human cyclin E2 (SEQ IDNO:8); this sequence identifies those codons that can be changed foroptimal expression of the DNA in both eukaryotic and prokaryotic hostcells. In this Figure, the letters B, D, H, K, M, R, S, V, W, Y, and Nhave the standard IUPAC meaning for nucleotides.

FIG. 12 is a bar graph of a Northern blot which quantitates mRNAexpression levels of human cyclin E2, human cyclin E1, and the enzymeGADPH (as a control) in various human tissues. The human tissues areindicated on the x-axis. The bar graph was generated by computer scan ofthe Northern blot after probing with each probe. Cyclin E1, cyclin E2,and GADPH are indicated.

FIG. 13 is a Northern blot of human breast tumor derived cell linesprobed with cyclin E1 and cyclin E2 probes. "Normal" refers to normalimmortalized cells. Estrogen receptor positive ("ER+") and estrogenreceptor negative ("ER-") tumor derived cell lines are indicated. Thename of each cell line is indicated at the top of the blot.

FIG. 14 depicts an "ambiguous sequence" of mouse cyclin E2 (SEQ IDNO:19); this sequence identifies those codons that can be changed foroptimal expression of the DNA in both eukaryotic and prokaryotic hostcells. The letters have the standard IUPAC meaning for nucleotides.

FIG. 15 depicts a Western blot (15A) and an kinase assay (15B) of ahuman osteosarcoma derived cell line termed Saos-2. In 15A, cells werelysed and immunoprecipitated with either pre-immune ("PI"; Lane 2) orwith anti-cyclin E2 antiserum ("E2"; Lane 3). The immunoprecipitateswere run on SDS-PAGE from which a Western blot was generated. The blotwas probed with an anti-cdk2 antibody. In 15B, extract was prepared fromthe same cell line and was treated with anti-cyclin E2 antibody (Lanes2-4) or preimmune serum (Lane 14), GST-Rb protein (Lane 3), or histoneH1 protein (Lane 2) was then added to the immunoprecipitate. After 30minutes at 37C., the immunoprecipitates were run on SDS-PAGE, andexposed to film for 2 hours.

DETAILED DESCRIPTION OF THE INVENTION

Included in the scope of this invention are cyclin E2 polypeptides suchas the polypeptides of SEQ ID NO:3 and SEQ ID NO:4, and relatedbiologically active polypeptide fragments, variants, and derivativesthereof. These cyclin E2 polypeptides share a homology of only about 47percent with cyclin E1 polypeptides at the amino acid level and about 55percent at the DNA level.

Included within the scope of the present invention are nucleic acidmolecules that encode cyclin E2 polypeptides, and methods for preparingthe polypeptides.

Also included within the scope of the present invention are non-humanmammals such as mice, rats, rabbits, goats, or sheep in which the gene(or genes) encoding native cyclin E2 has (have) been disrupted ("knockedout") such that the level of expression of this gene or genes is (are)significantly decreased or completely abolished. Such mammals may beprepared using techniques and methods such as those described in U.S.Pat. No. 5,557,032. The present invention further includes non-humanmammals such as mice, rats, rabbits, goats, or sheep in which the gene(or genes) encoding cyclin E2 (either the native form of cyclin E2 forthe mammal or a heterologous cyclin E2 gene(s)) is (are) over expressedby the mammal, thereby creating a "transgenic" mammal. Such transgenicmammals may be prepared using well known methods such as those describedin U.S. Pat. No 5,489,743 and PCT patent application no. W094/28122,published Dec. 8, 1994. The present invention further includes non-humanmammals in which the cyclin E2 promoter is either activated orinactivated (using homologous recombination methods as described below)to alter the level of expression of native cyclin E2.

The cyclin E2 polypeptides of the present invention may be added tocells to enhance cell division. Alternatively, cyclin E2 polypeptideactivity in a cell may be inhibited or inactivated in order to decreaseor stop cell division of certain cells.

The term "cyclin E2 protein" or "cyclin E2 polypeptide" as used hereinrefers to any protein or polypeptide having the properties describedherein for cyclin E2. The small letter in front of the term "cyclin E2",when used, refers to a cyclin E2 polypeptide from a particular mammal,i.e., "h-cyclin E2" refers to human cyclin E2, and "m-cyclin E2" refersto mouse cyclin E2. The cyclin E2 polypeptide may or may not have anamino terminal methionine, depending on the manner in which it isprepared. By way of illustration, cyclin E2 protein or cyclin E2polypeptide refers to (1) a biologically active polypeptide encoded bycyclin E2 nucleic acid molecules as defined in any of items (a)-(f)below, and biologically active peptide or polypeptide fragments thereof;(2) naturally occurring allelic variants and synthetic variants of thecyclin E2 gene which encode a cyclin E2 polypeptide that has one or moreamino acid substitutions deletions, and/or insertions as compared to thecyclin E2 polypeptide of SEQ ID NO:3 or SEQ ID NO:4, and/or (3)biologically active polypeptides, or fragments or variants thereof, thathave been chemically modified.

As used herein, the term "cyclin E2 fragment" refers to a peptide orpolypeptide that is less than the full length amino acid sequence ofnaturally occurring cyclin E2 protein but has cyclin E2 biologicalactivity. Such a fragment may be truncated at the amino terminus, thecarboxy terminus, and/or internally (such as by natural splicing), andmay be a variant or a derivative of cyclin E2. Such cyclin E2 fragmentsmay be prepared with or without an amino terminal methionine. Inaddition, cyclin E2 fragments can be naturally occurring fragments suchas the cyclin E2 splice variant (SEQ ID NO:5), other splice variants,and fragments resulting from in vivo protease activity.

As used herein, the term "cyclin E2 variant" refers to a cyclin E2polypeptide whose amino acid sequence contains one or more amino acidsequence substitutions, deletions, and/or insertions as compared to thecyclin E2 amino acid sequence set forth in SEQ ID NOS: 3 and 4. Suchcyclin E2 variants can be prepared from the corresponding cyclin E2nucleic acid molecule variants, which have a DNA sequence that variesaccordingly from the DNA sequences for wild type cyclin E2 as set forthin SEQ ID NOS: 1 and 2. Preferred variants of the human cyclin E2polypeptide include alanine substitutions at one or more of amino acidpositions 12-23, 32-53, 350-357, and 395-404 which can serve to alterthe substrate specificity of the cyclin E2 polypeptide. Other preferredvariants include alanine substitutions at amino acids positions 392,396, 397, and/or 401, which mutants can serve to increase the stabilityof the polypeptide.

As used herein, the term "cyclin E2 derivative" refers to a cyclin E2polypeptide, protein, or fragment that has been chemically modified, asfor example, by addition of one or more polyethylene glycol molecules,sugars, phosphates, and/or other such molecules, where the molecule ormolecules are not naturally attached to wild-type cyclin E2 polypeptide.

As used herein, the terms "biologically active cyclin E2 polypeptide","biologically active cyclin E2 fragment", "biologically active cyclin E2variant", and "biologically active cyclin E2 derivative" refer to acyclin E2 polypeptide that naturally forms a complex with cyclindependent kinase 2 ("cdk2") and is capable of phosphorylating theretinoblastoma ("Rb") gene product.

As used herein, the term "cyclin E2" when used to describe a nucleicacid molecule refers to a nucleic acid molecule or fragment thereof that(a) has the nucleotide sequence as set forth in SEQ ID NO:1 or SEQ IDNO:2; (b) has a nucleic acid sequence encoding a polypeptide that is atleast 70 percent identical, but may be greater than 70 percent, i.e.,75, 80, 85, 90, 95 percent, or even greater than 95 percent identical,to the polypeptide encoded by any of SEQ ID NOS:1 or 2; (c) is anaturally occurring allelic variant of (a) or (b); (d) is a nucleic acidvariant of (a)-(c) produced as provided for herein; (e) has a sequencethat is complementary to (a)-(d); and/or (f) hybridizes to any of(a)-(e) under conditions of high stringency.

Percent sequence identity can be determined by standard methods that arecommonly used to compare the similarity in position of the amino acidsof two polypeptides. By way of example, using a computer algorithm suchas BLAST, BLAST2, or FASTA, the two polypeptides for which the percentsequence identity is to be determined are aligned for optimal matchingof their respective amino acids (the "matched span", which can includethe full length of one or both sequences, or a pre-determined portion ofone or both sequences). Each computer program provides a "default"opening penalty and a "default" gap penalty, and a scoring matrix suchas PAM 250 (for FASTA) or BLOSUM 62 (for BLAST algorithms). A preferredalgorithm is BLAST2.

A standard scoring matrix (see Dayhoff et al., in: Atlas of ProteinSequence and Structure, vol. 5, supp.3 [1978]) can be used inconjunction with the computer algorithm. The percent identity can thenbe calculated by determining the percent identity using an algorithmcontained in a program such as FASTA: ##EQU1## Polypeptides that are atleast 70 percent identical will typically have one or more amino acidsubstitutions, deletions, and/or insertions as compared with wild typecyclin E2. Usually, the substitutions of the native residue will beeither alanine, or a conservative amino acid so as to have little or noeffect on the overall net charge, polarity, or hydrophobicity of theprotein. Conservative substitutions are set forth in Table I below.

                  TABLE I                                                         ______________________________________                                        Conservative Amino Acid Substitutions                                         ______________________________________                                        Basic:               arginine                                                    lysine                                                                        histidine                                                                    Acidic: glutamic acid                                                          aspartic acid                                                                Uncharged Polar: glutamine                                                     asparagine                                                                    serine                                                                        threonine                                                                     tyrosine                                                                     Non-Polar: phenylalanine                                                       tryptophan                                                                    cysteine                                                                      glycine                                                                       alanine                                                                       valine                                                                        proline                                                                       methionine                                                                    leucine                                                                       isoleucine                                                                 ______________________________________                                    

The term "conditions of high stringency" refers to hybridization andwashing under conditions that permit binding of a nucleic acid moleculeused for screening, such as an oligonucleotide probe or cDNA moleculeprobe, to highly homologous sequences. An exemplary high stringency washsolution is 0.2×SSC and 0.1 percent SDS used at a temperature of between50° C.-65° C.

Where oligonucleotide probes are used to screen cDNA or genomiclibraries, one of the following two high stringency solution may beused. The first of these is 6×SSC with 0.05 percent sodium pyrophosphateat a temperature of 35° C.-62° C., depending on the length of theoligonucleotide probe. For example, 14 base pair probes are washed at35° C.-40° C., 17 base pair probes are washed at 45° C.-50° C., 20 basepair probes are washed at 52° C.-57° C., and 23 base pair probes arewashed at 57-63° C. The temperature can be increased 2-3° C. where thebackground non-specific binding appears high. A second high stringencysolution utilizes tetramethylammonium chloride (TMAC) for washingoligonucleotide probes. One stringent washing solution is 3 M TMAC, 50mM Tris-HCl, pH 8.0, and 0.2 percent SDS. The washing temperature usingthis solution is a function of the length of the probe. For example, a17 base pair probe is washed at about 45-50° C.

As used herein, the terms "effective amount" and "therapeuticallyeffective amount" refer to the amount of cyclin E2 necessary to supportone or more biological activities of cyclin E2 as set forth above.

A full length cyclin E2 polypeptide or fragment thereof can be preparedusing well known recombinant DNA technology methods such as those setforth in Sambrook et al. (Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1989]) and/orAusubel et al., eds, (Current Protocols in Molecular Biology, GreenPublishers Inc. and Wiley and Sons, N.Y. [1994]). A gene or cDNAencoding a cyclin E2 protein or fragment thereof may be obtained forexample by screening a genomic or cDNA library, or by PCR amplification.Probes or primers useful for screening the library can be generatedbased on sequence information for other known genes or gene fragmentsfrom the same or a related family of genes, such as, for example,conserved motifs found in other cyclin genes such as the cyclin box. Inaddition, where a cyclin gene has been identified from one species, allor a portion of that gene may be used as a probe to identify homologousgenes from other species. The probes or primers may be used to screencDNA libraries from various tissue sources believed to express thecyclin E2 gene. Typically, conditions of high stringency will beemployed for screening to minimize the number of false positivesobtained from the screen.

Another means to prepare a gene encoding cyclin E2 polypeptide orfragment thereof is to employ chemical synthesis using methods wellknown to the skilled artisan such as those described by Engels et al.(Angew. Chem. Intl. Ed., 28:716-734 [1989]). These methods include,inter alia, the phosphotriester, phosphoramidite, and H-phosphonatemethods for nucleic acid synthesis. A preferred method for such chemicalsynthesis is polymer-supported synthesis using standard phosphoramiditechemistry. Typically, the DNA encoding the cyclin E2 polypeptide will beseveral hundred nucleotides in length. Nucleic acids larger than about100 nucleotides can be synthesized as several fragments using thesemethods. The fragments can then be ligated together to form the fulllength cyclin E2 polypeptide. Usually, the DNA fragment encoding theamino terminus of the polypeptide will have an ATG, which encodes amethionine residue. This methionine may or may not be present on themature form of the cyclin E2 polypeptide, depending on whether thepolypeptide produced in the host cell is secreted from that cell.

In some cases, it may be desirable to prepare nucleic acid and/or aminoacid variants of naturally occurring cyclin E2. Nucleic acid variants(wherein one or more nucleotides are designed to differ from thewild-type or naturally occurring cyclin E2) may be produced using sitedirected mutagenesis, PCR amplification, or other appropriate methods,where the primer(s) have the desired point mutations (see Sambrook etal., supra, and Ausubel et al., supra, for descriptions of mutagenesistechniques). Chemical synthesis using methods described by Engels etal., supra, may also be used to prepare such variants. Other methodsknown to the skilled artisan may be used as well. Preferred nucleic acidvariants are those containing nucleotide substitutions accounting forcodon preference in the host cell that is to be used to produce cyclinE2. Other preferred variants are those encoding conservative amino acidchanges as described above (e.g., wherein the charge or polarity of thenaturally occurring amino acid side chain is not altered substantiallyby substitution with a different amino acid) as compared to wild type,and/or those designed to either generate a novel glycosylation and/orphosphorylation site(s) on cyclin E2, or those designed to delete anexisting glycosylation and/or phosphorylation site(s) on cyclin E2.

The cyclin E2 gene, cDNA, or fragment thereof can be inserted into anappropriate expression or amplification vector using standard ligationtechniques. The vector is typically selected to be functional in theparticular host cell employed (i.e., the vector is compatible with thehost cell machinery such that amplification of the cyclin E2 gene and/orexpression of the gene can occur). The cyclin E2 gene, cDNA or fragmentthereof may be amplified/expressed in prokaryotic, yeast, insect(baculovirus systems) and/or eukaryotic host cells. Selection of thehost cell will depend in part on whether the cyclin E2 polypeptide orfragment thereof is to be glycosylated and/or phosphorylated. If so,yeast, insect, or mammalian host cells are preferable.

Typically, the vectors used in any of the host cells will contain 5'flanking sequence (also referred to as a "promoter") and otherregulatory elements as well such as an enhancer(s), an origin ofreplication element, a transcriptional termination element, a completeintron sequence containing a donor and acceptor splice site, a signalpeptide sequence, a ribosome binding site element, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these elements is discussed below. Optionally, the vector may containa "tag" sequence, i.e., an oligonucleotide molecule located at the 5' or3' end of the cyclin E2 coding sequence; the oligonucleotide moleculeencodes polyHis (such as hexaHis), or other "tag" such as FLAG, HA(hemaglutinin Influenza virus) or myc for which commercially availableantibodies exist. This tag is typically fused to the protein, and canserve as an tag for affinity purification of the cyclin E2 polypeptidefrom the host cell. Affinity purification can be accomplished, forexample, by column chromatography using antibodies against the tag as anaffinity matrix. Optionally, the tag can subsequently be removed fromthe purified cyclin E2 polypeptide by various means such as usingcertain peptidases.

The 5' flanking sequence may be homologous (i.e., from the same speciesand/or strain as the host cell), heterologous (i.e., from a speciesother than the host cell species or strain), hybrid (i.e., a combinationof 5' flanking sequences from more than one source), synthetic, or itmay be the native cyclin E2 5' flanking sequence. As such, the source ofthe 5' flanking sequence may be any unicellular prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the 5' flanking sequence is functional in, and canbe activated by, the host cell machinery.

The 5' flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically, 5'flanking sequences useful herein other than the cyclin E2 flankingsequence will have been previously identified by mapping and/or byrestriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full nucleotide sequence of the 5' flanking sequence maybe known. Here, the 5' flanking sequence may be synthesized using themethods described above for nucleic acid synthesis or cloning.

Where all or only a portion of the 5' flanking sequence is known, it maybe obtained using PCR and/or by screening a genomic library withsuitable oligonucleotide and/or 5' flanking sequence fragments from thesame or another species.

Where the 5' flanking sequence is not known, a fragment of DNAcontaining a 5' flanking sequence may be isolated from a larger piece ofDNA that may contain, for example, a coding sequence or even anothergene or genes. Isolation may be accomplished by restriction endonucleasedigestion using one or more carefully selected enzymes to isolate theproper DNA fragment. After digestion, the desired fragment may beisolated by agarose gel purification, Qiagen® column or other methodsknown to the skilled artisan. Selection of suitable enzymes toaccomplish this purpose will be readily apparent to one of ordinaryskill in the art.

The origin of replication element is typically a part of prokaryoticexpression vectors purchased commercially, and aids in the amplificationof the vector in a host cell. Amplification of the vector to a certaincopy number can, in some cases, be important for optimal expression ofthe cyclin E2 polypeptide. If the vector of choice does not contain anorigin of replication site, one may be chemically synthesized based on aknown sequence, and ligated into the vector.

The transcription termination element is typically located 3' of the endof the cyclin E2 polypeptide coding sequence and serves to terminatetranscription of the cyclin E2 polypeptide. Usually, the transcriptiontermination element in prokaryotic cells is a G-C rich fragment followedby a poly T sequence. While the element is easily cloned from a libraryor even purchased commercially as part of a vector, it can also bereadily synthesized using methods for nucleic acid synthesis such asthose described above.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene.

The ribosome binding element, commonly called the Shine-Dalgarnosequence (prokaryotes) or the Kozak sequence (eukaryotes), is usuallynecessary for translation initiation of mRNA. The element is typicallylocated 3' to the promoter and 5' to the coding sequence of the cyclinE2 polypeptide to be synthesized. The Shine-Dalgarno sequence is variedbut is typically a polypurine (i.e., having a high A-G content). ManyShine-Dalgarno sequences have been identified, each of which can bereadily synthesized using methods set forth above and used in aprokaryotic vector.

In those cases where it is desirable for cyclin E2 polypeptide to besecreted from the host cell, a signal sequence may be used to direct thecyclin E2 polypeptide out of the host cell where it is synthesized, andthe carboxy-terminal part of the protein may be deleted in order toprevent membrane anchoring. Typically, the signal sequence is positionedin the coding region of the cyclin E2 nucleic acid sequence, or directlyat the 5' end of the cyclin E2 coding region. Many signal sequences havebeen identified, and any of them that are functional in the selectedhost cell may be used in conjunction with the cyclin E2 gene. Therefore,the signal sequence may be homologous or heterologous to the cyclin E2gene, and may be homologous or heterologous to the cyclin E2 gene.Additionally, the signal sequence may be chemically synthesized usingmethods set forth above.

In most cases, secretion of the polypeptide from the host cell via thepresence of a signal peptide will result in the removal of the aminoterminal methionine from the polypeptide.

In many cases, transcription of the cyclin E2 gene is increased by thepresence of one or more introns in the vector; this is particularly truewhere cyclin E2 is produced in eukaryotic host cells, especiallymammalian host cells. The introns used may be naturally occurring withinthe cyclin E2 gene, especially where the cyclin E2 gene used is a fulllength genomic sequence or a fragment thereof. Where the intron is notnaturally occurring within the cyclin E2 gene (as for most cDNAS), theintron(s) may be obtained from another source. The position of theintron with respect to the 5' flanking sequence and the cyclin E2 geneis generally important, as the intron must be transcribed to beeffective. As such, where the cyclin E2 gene inserted into theexpression vector is a cDNA molecule, the preferred position for theintron is 3' to the transcription start site, and 5' to the polyAtranscription termination sequence. Preferably for cyclin E2 cDNA, theintron will be located on one side or the other (i.e., 5' or 3') of thecDNA such that it does not interrupt the this coding sequence. Anyintron from any source, including any viral, prokaryotic and eukaryotic(plant or animal) organisms, may be used to practice this invention,provided that it is compatible with the host cell(s) into which it isinserted. Also included herein are synthetic introns. Optionally, morethan one intron may be used in the vector.

Where one or more of the elements set forth above are not alreadypresent in the vector to be used, they may be individually obtained andligated into the vector. Methods used for obtaining each of the elementsare well known to the skilled artisan and are comparable to the methodsset forth above (i.e., synthesis of the DNA, library screening, and thelike).

The final vectors used to practice this invention are typicallyconstructed from a starting vectors such as a commercially availablevector. Such vectors may or may not contain some of the elements to beincluded in the completed vector. If none of the desired elements arepresent in the starting vector, each element may be individually ligatedinto the vector by cutting the vector with the appropriate restrictionendonuclease(s) such that the ends of the element to be ligated in andthe ends of the vector are compatible for ligation. In some cases, itmay be necessary to "blunt" the ends to be ligated together in order toobtain a satisfactory ligation. Blunting is accomplished by firstfilling in "sticky ends" using Klenow DNA polymerase or T4 DNApolymerase in the presence of all four nucleotides. This procedure iswell known in the art and is described for example in Sambrook et al.,supra.

Alternatively, two or more of the elements to be inserted into thevector may first be ligated together (if they are to be positionedadjacent to each other) and then ligated into the vector.

One other method for constructing the vector to conduct all ligations ofthe various elements simultaneously in one reaction mixture. Here, manynonsense or nonfunctional vectors will be generated due to improperligation or insertion of the elements, however the functional vector maybe identified and selected by restriction endonuclease digestion.

Preferred vectors for practicing this invention are those which arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3 (InvitrogenCompany, San Diego, Calif.), pBSII (Stratagene Company, LaJolla,Calif.), pET15b (Novagen, Madison, Wis.), PGEX (Pharmacia Biotech,Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), PETL(BlueBacII; Invitrogen), and pFastBacDual (Gibco/BRL, Grand Island,N.Y.).

After the vector has been constructed and a nucleic acid moleculeencoding full length or truncated cyclin E2 has been inserted into theproper site of the vector, the completed vector may be inserted into asuitable host cell for amplification and/or cyclin E2 polypeptideexpression.

Host cells may be prokaryotic host cells (such as E. coli) or eukaryotichost cells (such as a yeast cell, an insect cell, or a vertebrate cell).The host cell, when cultured under appropriate conditions, cansynthesize cyclin E2 polypeptide which can subsequently be collectedfrom the culture medium (if the host cell secretes it into the medium)or directly from the host cell producing it (if it is not secreted).After collection, the cyclin E2 polypeptide can be purified usingmethods such as molecular sieve chromatography, affinity chromatography,and the like.

Selection of the host cell for cyclin E2 polypeptide production willdepend in part on whether the cyclin E2 polypeptide is to beglycosylated or phosphorylated (in which case eukaryotic host cells arepreferred), and the manner in which the host cell is able to "fold" theprotein into its native tertiary structure (e.g., proper orientation ofdisulfide bridges, etc.) such that biologically active protein isprepared by the cell. However, where the host cell does not synthesizecyclin E2 polypeptide that has biological activity, the cyclin E2polypeptide may be "folded" after synthesis using appropriate chemicalconditions as discussed below.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO) or 3T3 cells. The selection of suitablemammalian host cells and methods for transformation, culture,amplification, screening and product production and purification areknown in the art. Other suitable mammalian cell lines, are the monkeyCOS-1 and COS-7 cell lines, and the CV-1 cell line. Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Candidate cells may be genotypicallydeficient in the selection gene, or may contain a dominantly actingselection gene. Other suitable mammalian cell lines include but are notlimited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster celllines.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, DH5α,DH10, and MC1061) are well-known as host cells in the fieldof biotechnology. Various strains of B. subtilis, Pseudomonas spp.,other Bacillus spp., Streptomyces spp., and the like may also beemployed in this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for expression of the polypeptides of thepresent invention.

Additionally, where desired, insect cell systems may be utilized in themethods of the present invention. Such systems are described for examplein Kitts et al. (Biotechniques, 14:810-817 [1993]), Lucklow (Curr. Opin.Biotechnol., 4:564-572 [1993]) and Lucklow et al. (J. Virol.,67:4566-4579 [1993]). Preferred insect cells are Sf-9 and Hi5(Invitrogen, Carlsbad, Calif.).

Insertion (also referred to as "transformation" or "transfection") ofthe vector into the selected host cell may be accomplished using suchmethods as calcium chloride, electroporation, microinjection,lipofection or the DEAE-dextran method. The method selected will in partbe a function of the type of host cell to be used. These methods andother suitable methods are well known to the skilled artisan, and areset forth, for example, in Sambrook et al., supra.

The host cells containing the vector (i.e., transformed or transfected)may be cultured using standard media well known to the skilled artisan.The media will usually contain all nutrients necessary for the growthand survival of the cells. Suitable media for culturing E. coli cellsare for example, Luria Broth (LB) and/or Terrific Broth (TB). Suitablemedia for culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all ofwhich may be supplemented with serum and/or growth factors as requiredby the particular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate, and/or fetal calf serum as necessary.

Typically, an antibiotic or other compound useful for selective growthof the transformed cells only is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent on the plasmid with which the host cell was transformed. Forexample, where the selectable marker element is kanamycin resistance,the compound added to the culture medium will be kanamycin.

The amount of cyclin E2 polypeptide produced in the host cell can beevaluated using standard methods known in the art. Such methods include,without limitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, HPLC separation,immunoprecipitation, and/or activity assays such as DNA binding gelshift assays.

If the cyclin E2 polypeptide has been designed to be secreted from thehost cells, the majority of polypeptide may be found in the cell culturemedium. Polypeptides prepared in this way will typically not possess anamino terminal methionine, as it is removed during secretion from thecell. If however, the cyclin E2 polypeptide is not secreted from thehost cells, it will be present in the cytoplasm and/or the nucleus (foreukaryotic host cells) or in the periplasm (for gram negative bacteriahost cells) and may have an amino terminal methionine.

For cyclin E2 polypeptide situated in the host cell cytoplasm and/ornucleus, the host cells are typically first disrupted mechanically orwith detergent to release the intra-cellular contents into a bufferedsolution. Cyclin E2 polypeptide can then be isolated from this solution.

Purification of cyclin E2 polypeptide from solution can be accomplishedusing a variety of techniques. If the polypeptide has been synthesizedsuch that it contains a tag such as Hexahistidine (cyclin E2/hexaHis) orother small peptide such as FLAG (Eastman Kodak Co., New Haven, Conn.)or myc (Invitrogen, Carlsbad, Calif.) at either its carboxyl or aminoterminus, it may essentially be purified in a one-step process bypassing the solution through an affinity column where the column matrixhas a high affinity for the tag or for the polypeptide directly (i.e., amonoclonal antibody specifically recognizing cyclin E2). For example,polyhistidine binds with great affinity and specificity to nickel, thusan affinity column of nickel (such as the Qiagen® nickel columns) can beused for purification of cyclin E2/polyHis. (See for example, Ausubel etal., eds., Current Protocols in Molecular Biology, Section 10.11.8, JohnWiley & Sons, New York [1993]).

Where the cyclin E2 polypeptide is prepared without a tag attached, andno antibodies are available, other well known procedures forpurification can be used. Such procedures include, without limitation,ion exchange chromatography, molecular sieve chromatography, HPLC,native gel electrophoresis in combination with gel elution, andpreparative isoelectric focusing ("Isoprime" machine/technique, HoeferScientific). In some cases, two or more of these techniques may becombined to achieve increased purity.

If it is anticipated that the cyclin E2 polypeptide will be foundprimarily intracellularly, the intracellular material (includinginclusion bodies for gram-negative bacteria) can be extracted from thehost cell using any standard technique known to the skilled artisan. Forexample, the host cells can be lysed to release the contents of theperiplasm/cytoplasm by French press, homogenization, and/or sonicationfollowed by centrifugation.

If the cyclin E2 polypeptide has formed inclusion bodies in theperiplasm, the inclusion bodies can often bind to the inner and/or outercellular membranes and thus will be found primarily in the pelletmaterial after centrifugation. The pellet material can then be treatedwith a chaotropic agent such as guanidine or urea to release, breakapart, and solubilize the inclusion bodies. The cyclin E2 polypeptide inits now soluble form can then be analyzed using gel electrophoresis,immunoprecipitation or the like. If it is desired to isolate the cyclinE2 polypeptide, isolation may be accomplished using standard methodssuch as those set forth below and in Marston et al. (Meth. Enz.,182:264-275 [1990]).

If cyclin E2 polypeptide inclusion bodies are not formed to asignificant degree in the periplasm of the host cell, the cyclin E2polypeptide will be found primarily in the supernatant aftercentrifugation of the cell homogenate, and the cyclin E2 polypeptide canbe isolated from the supernatant using methods such as those set forthbelow.

In those situations where it is preferable to partially or completelyisolate the cyclin E2 polypeptide, purification can be accomplishedusing standard methods well known to the skilled artisan. Such methodsinclude, without limitation, separation by electrophoresis followed byelectroelution, various types of chromatography (immunoaffinity,molecular sieve, and/or ion exchange), and/or high pressure liquidchromatography. In some cases, it may be preferable to use more than oneof these methods for complete purification.

In addition to preparing and purifying cyclin E2 polypeptide usingrecombinant DNA techniques, the cyclin E2 polypeptides, fragments,and/or derivatives thereof may be prepared by chemical synthesis methods(such as solid phase peptide synthesis) using techniques known in theart such as those set forth by Merrifield et al., (J. Am. Chem. Soc.,85:2149 [1963]), Houghten et al. (Proc Natl Acad. Sci. USA, 82:5132[1985]), and Stewart and Young (Solid Phase Peptide Synthesis, PierceChemical Co., Rockford, Ill. [1984]). Such polypeptides may besynthesized with or without a methionine on the amino terminus.Chemically synthesized cyclin E2 polypeptides or fragments may beoxidized using methods set forth in these references to form disulfidebridges. The cyclin E2 polypeptides or fragments are expected to havebiological activity comparable to cyclin E2 polypeptides producedrecombinantly or purified from natural sources, and thus may be usedinterchangably with recombinant or natural cyclin E2 polypeptide.

Chemically modified cyclin E2 compositions in which cyclin E2polypeptide is linked to a polymer are included within the scope of thepresent invention. The polymer selected is typically water soluble sothat the protein to which it is attached does not precipitate in anaqueous environment, such as a physiological environment. The polymerselected is usually modified to have a single reactive group, such as anactive ester for acylation or an aldehyde for alkylation, so that thedegree of polymerization may be controlled as provided for in thepresent methods. The polymer may be of any molecular weight, and may bebranched or unbranched. Included within the scope of cyclin E2 polymersis a mixture of polymers. Preferably, for therapeutic use of theend-product preparation, the polymer will be pharmaceuticallyacceptable.

The water soluble polymer or mixture thereof may be selected from thegroup consisting of, for example, polyethylene glycol (PEG),monomethoxypolyethylene glycol, dextran, cellulose, or othercarbohydrate based polymers, poly-(N-vinyl pyrrolidone)polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol.

For the acylation reactions, the polymer(s) selected should have asingle reactive ester group. For reductive alkylation, the polymer(s)selected should have a single reactive aldehyde group. A preferredreactive aldehyde is polyethylene glycol propionaldehyde, which is waterstable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see U.S.Pat. No. 5,252,714).

Pegylation of cyclin E2 may be carried out by any of the pegylationreactions known in the art, as described for example in the followingreferences: Focus on Growth Factors 3: 4-10 (1992); EP 0 154 316; and EP0 401 384. Preferably, the pegylation is carried out via an acylationreaction or an alkylation reaction with a reactive polyethylene glycolmolecule (or an analogous reactive water-soluble polymer) as describedbelow.

A particularly preferred water-soluble polymer for use herein ispolyethylene glycol, abbreviated PEG. As used herein, polyethyleneglycol is meant to encompass any of the forms of PEG that have been usedto derivatize other proteins, such as mono-(C1-C10) alkoxy- oraryloxy-polyethylene glycol.

In general, chemical derivatization may be performed under any suitableconditions used to react a biologically active substance with anactivated polymer molecule. Methods for preparing pegylated cyclin E2will generally comprise the steps of (a) reacting a cyclin E2polypeptide with polyethylene glycol (such as a reactive ester oraldehyde derivative of PEG) under conditions whereby cyclin E2 becomesattached to one or more PEG groups, and (b) obtaining the reactionproduct(s). In general, the optimal reaction conditions for theacylation reactions will be determined based on known parameters and thedesired result. For example, the larger the ratio of PEG: protein, thegreater the percentage of poly-pegylated product.

Generally, conditions which may be alleviated or modulated byadministration of the present polymer/cyclin E2 polypeptides includethose described herein for cyclin E2 molecules. However, thepolymer/cyclin E2 molecules disclosed herein may have additionalactivities, enhanced or reduced biological activity, or othercharacteristics, such as increased or decreased half-life, as comparedto the non-derivatized molecules.

The cyclin E2 polypeptides, fragments thereof, variants, andderivatives, may be employed alone, together, or in combination withother pharmaceutical compositions. The cyclin E2 polypeptides,fragments, variants, and derivatives may be used in combination withcytokines, growth factors, antibiotics, anti-inflammatories, and/orchemotherapeutic agents as is appropriate for the indication beingtreated.

The cyclin E2 molecules, whether administered alone or in combinationtherapy, may be useful in promoting cell division of particular cellpopulations such as white blood cells, red blood cells, neurons,chondrocytes, and the like. In AIDS patients and cancer patients thathave undergone chemotherapy and/or bone marrow transplants, the whiteblood cell count is typically low; administration of cyclin E2 to thepatient's stem cells and/or other white blood cells, either by genetherapy or by direct treatment of the cells with cyclin E2 in an ex vivomanner, could serve to increase the white blood cell count. Similarly,in hemophiliacs and kidney dialysis patients, for example, treatment ofthe patient's progenitor erythroblast cell population with cyclin E2 viagene therapy or ex vivo treatment, could enhance red blood cell count.Another indication in which cyclin E2 administration could be useful isto increase chondrocytes in patients suffering from degeneration ofcartilage due to joint injury, arthritis, or the like. Here, cyclin E2could be administered to chondrocytes either via gene therapy or by exvivo treatment of cultured chondrocytes. Still another indication forcyclin E2 therapy would be to expand the population of neurons inpatients suffering from Alzheimer's disease, or other neurologicaldisorders that result from apoptosis of various neurons. Finally, cyclinE2 therapy could be indicated in stroke or ischemia in which tissuedamage results from loss of blood flow. Here, administration of cyclinE2 to cells surrounding the area of necrosis could serve to regeneratethe necrotic tissue.

Cyclin E2 nucleic acid molecules, fragments, and/or derivatives that donot themselves encode polypeptides that are active in activity assaysmay be useful as hybridization probes in diagnostic assays to test,either qualitatively or quantitatively, for the presence of cyclin E2DNA or corresponding RNA in mammalian tissue or bodily fluid samples.

Cyclin E2 polypeptide fragments, variants, and/or derivatives that arenot themselves active in activity assays may be useful for preparingantibodies that recognize cyclin E2 polypeptides.

The cyclin E2 polypeptides, fragments, variants, and/or derivatives maybe used to prepare antibodies using standard methods. Thus, antibodiesthat react with the cyclin E2 polypeptides, as well as reactivefragments of such antibodies, are also contemplated as within the scopeof the present invention. The antibodies may be polyclonal, monoclonal,recombinant, chimeric, single-chain and/or bispecific. Typically, theantibody or fragment thereof will either be of human origin, or will be"humanized", i.e., prepared so as to prevent or minimize an immunereaction to the antibody when administered to a patient. The antibodyfragment may be any fragment that is reactive with the cyclin E2polypeptides of the present invention, such as, F_(ab), F_(ab'), etc.Also provided by this invention are the hybridomas generated bypresenting cyclin E2 or a fragment thereof as an antigen to a selectedmammal, followed by fusing cells (e.g., spleen cells) of the mammal withcertain cancer cells to create immortalized cell lines by knowntechniques. The methods employed to generate such cell lines andantibodies directed against all or portions of a human cyclin E2polypeptide of the present invention are also encompassed by thisinvention.

The antibodies may be used therapeutically, such as to inhibit bindingof cyclin E2 to cdk2. The antibodies may further be used for in vivo andin vitro diagnostic purposes, such as in labeled form to detect thepresence of cyclin E2 in a body fluid or cell sample.

Preferred antibodies are human antibodies, either polyclonal ormonoclonal.

Therapeutic Compositions and Administration

Therapeutic compositions of cyclin E2 are within the scope of thepresent invention. Such compositions may comprise a therapeuticallyeffective amount of cyclin polypeptide, fragments, variants, orderivatives in admixture with a pharmaceutically acceptable carrier. Thecarrier material may be water for injection, preferably supplementedwith other materials common in solutions for administration to mammals.Typically, a cyclin E2 therapeutic compound will be administered in theform of a composition comprising purified cyclin E2 polypeptide,fragment, variant, or derivative in conjunction with one or morephysiologically acceptable carriers, excipients, or diluents. Neutralbuffered saline or saline mixed with serum albumin are exemplaryappropriate carriers. Preferably, the product is formulated as alyophilizate using appropriate excipients (e.g., sucrose). Otherstandard carriers, diluents, and excipients may be included as desired.Other exemplary compositions comprise Tris buffer of about pH 7.0-8.5,or acetate buffer of about pH 4.0-5.5, which may further includesorbitol or a suitable substitute therefor.

The cyclin E2 compositions can be administered parenterally.Alternatively, the compositions may be administered intravenously orsubcutaneously. When systemically administered, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such pharmaceutically acceptable protein solutions, with due regardto pH, isotonicity, stability and the like, is within the skill of theart.

Therapeutic formulations of cyclin E2 compositions useful for practicingthe present invention may be prepared for storage by mixing the selectedcomposition having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed.,Mack Publishing Company [1990]) in the form of a lyophilized cake or anaqueous solution. Acceptable carriers, excipients or stabilizers arenontoxic to recipients and are preferably inert at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,or other organic acids; antioxidants such as ascorbic acid; lowmolecular weight polypeptides; proteins, such as serum albumin, gelatin,or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, pluronics orpolyethylene glycol (PEG).

An effective amount of the cyclin E2 composition(s) to be employedtherapeutically will depend, for example, upon the therapeuticobjectives such as the indication for which cyclin E2 is being used, theroute of administration, and the condition of the patient. Accordingly,it will be necessary for the therapist to titer the dosage and modifythe route of administration as required to obtain the optimaltherapeutic effect. A typical daily dosage may range from about 0.1μg/kg to up to 100 mg/kg or more, depending on the factors mentionedabove. Typically, a clinician will administer the cyclin E2 compositionuntil a dosage is reached that achieves the desired effect. The cyclinE2 composition may therefore be administered as a single dose, or as twoor more doses (which may or may not contain the same amount of cyclinE2) over time, or as a continuous infusion via implantation device orcatheter.

As further studies are conducted, information will emerge regardingappropriate dosage levels for treatment of various conditions in variouspatients, and the ordinary skilled worker, considering the therapeuticcontext, the type of disorder under treatment, the age and generalhealth of the recipient, will be able to ascertain proper dosing.

The cyclin E2 composition to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes. Where the cyclin E2 composition is lyophilized,sterilization using these methods may be conducted either prior to, orfollowing, lyophilization and reconstitution. The composition forparenteral administration ordinarily will be stored in lyophilized formor in solution.

Therapeutic compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

The route of administration of the composition is in accord with knownmethods, e.g. oral, injection or infusion by intravenous,intraperitoneal, intracerebral (intraparenchymal),intracerebroventricular, intramuscular, intraocular, intraarterial, orintralesional routes, or by sustained release systems or implantationdevice which may optionally involve the use of a catheter. Wheredesired, the compositions may be administered continuously by infusion,bolus injection or by implantation device.

Alternatively or additionally, cyclin E2 may be administered locally viaimplantation into the affected area of a membrane, sponge, or otherappropriate material on to which cyclin E2 polypeptide has beenabsorbed.

Where an implantation device is used, the device may be implanted intoany suitable tissue or organ, and delivery of cyclin E2 may be directlythrough the device via bolus, or via continuous administration, or viacatheter using continuous infusion.

Cyclin E2 polypeptide may be administered in a sustained releaseformulation or preparation. Suitable examples of sustained-releasepreparations include semipermeable polymer matrices in the form ofshaped articles, e.g. films, or microcapsules. Sustained releasematrices include polyesters, hydrogels, polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al, Biopolymers, 22: 547-556 [1983]), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.,15: 167-277 [1981] and Langer, Chem. Tech., 12: 98-105 [1982]), ethylenevinyl acetate (Langer et al., supra) or poly-D(-)-3-hydroxybutyric acid(EP 133,988). Sustained-release compositions also may include liposomes,which can be prepared by any of several methods known in the art (e.g.,Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 [1985]; EP36,676; EP 88,046; EP 143,949).

In some cases, it may be desirable to use cyclin E2 compositions in anex vivo manner. Here, cells, tissues, or organs that have been removedfrom the patient are exposed to cyclin E2 compositions after which thecells, tissues and/or organs are subsequently implanted back into thepatient.

In other cases, cyclin E2 may be delivered through implanting intopatients certain cells that have been genetically engineered, usingmethods such as those described herein, to express and secrete cyclin E2polypeptides, fragments, variants, or derivatives. Such cells may beanimal or human cells, and may be derived from the patient's own tissueor from another source, either human or non-human. Optionally, the cellsmay be immortalized. However, in order to decrease the chance of animmunological response, it is preferred that the cells be encapsulatedto avoid infiltration of surrounding tissues. The encapsulationmaterials are typically biocompatible, semi-permeable polymericenclosures or membranes that allow release of the protein product(s) butprevent destruction of the cells by the patient's immune system or byother detrimental factors from the surrounding tissues.

Methods used for membrane encapsulation of cells are familiar to theskilled artisan, and preparation of encapsulated cells and theirimplantation in patients may be accomplished without undueexperimentation. See, e.g., U.S. Pat. Nos. 4,892,538; 5,011,472; and5,106,627. A system for encapsulating living cells is described in PCTWO 91/10425 (Aebischer et al.). Techniques for formulating a variety ofother sustained or controlled delivery means, such as liposome carriers,bio-erodible particles or beads, are also known to those in the art, andare described, for example, in U.S. Pat. No. 5,653,975 (Baetge et al.,CytoTherapeutics, Inc.). The cells, with or without encapsulation, maybe implanted into suitable body tissues or organs of the patient.

As discussed above, it may be desirable to treat isolated cellpopulations such as stem cells, lymphocytes, red blood cells,chondrocytes, neurons, and the like with cyclin E2. This can beaccomplished by exposing the isolated cells to cyclin E2 proteindirectly, where the cyclin E2 is in a form that is permeable to the cellmembrane. Alternatively, gene therapy can be employed as describedbelow.

One manner in which gene therapy can be applied is to use the cyclin E2gene (either genomic DNA, cDNA, and/or synthetic DNA encoding cyclin E2,or a fragment, variant, or derivative thereof) which may be operablylinked to a constitutive or inducible promoter to form a "gene therapyDNA construct". The promoter may be homologous or heterologous to theendogenous cyclin E2 gene, provided that it is active in the cell ortissue type into which the construct will be inserted. Other componentsof the gene therapy DNA construct may optionally include, as required,DNA molecules designed for site-specific integration (e.g., endogenousflanking sequences useful for homologous recombination), tissue-specificpromoter enhancers or silencers, DNA molecules capable of providing aselective advantage over the parent cell, DNA molecules useful as labelsto identify transformed cells, negative selection systems, cell specificbinding agents (as, for example, for cell targeting) cell-specificinternalization factors, and transcription factors to enhance expressionby a vector as well as factors to enable vector manufacture.

This gene therapy DNA construct can then be introduced into thepatient's cells (either ex vivo or in vivo). One means for introducingthe gene therapy DNA construct is via viral vectors. Suitable viralvectors typically used in gene therapy for delivery of gene therapy DNAconstructs include, without limitation, adenovirus, adeno-assoicatedvirus, herpes simplex virus, lentivirus, papilloma virus, and retrovirusvectors. Some of these vectors, such as retroviral vectors, will deliverthe gene therapy DNA construct to the chromosomal DNA of the patient'scells, and the gene therapy DNA construct can integrate into thechromosomal DNA; other vectors will function as episomes and the genetherapy DNA construct will remain in the cytoplasm. The use of genetherapy vectors is described, for example, in U.S. Pat. No. 5,672,344(Sep. 30, 1997; Kelly et al., University of Michigan), U.S. Pat. No.5,399,346 (Mar. 21, 1995; Anderson et al., U.S. Dept. Health and HumanServices), U.S. Pat. No. 5,631,236 (May 20, 1997; Woo et al., BaylorCollege of Medicine), and U.S. Pat. No. 5,635,399 (Jun. 3, 1997;Kriegler et al., Chiron Corp.).

Alternative means to deliver gene therapy DNA constructs to a patient'scells without the use of viral vectors include, without limitation,liposome-mediated transfer, direct injection of naked DNA,receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation, and microparticle bombardment (e.g.,"gene gun"). See U.S. Pat. No. 4,970,154 (Nov. 13, 1990; Chang, BaylorCollege of Medicine), WO 96/40958 (Dec. 19, 1996; Smith et al., BaylorCollege of Medicine) U.S. Pat. No. 5,679,559 (Oct. 21, 1997; Kim et al.,University of Utah) U.S. Pat. No. 5,676,954 (Oct. 14, 1997; Brigham,Vanderbilt University), and U.S. Pat. No. 5,593,875 (Jan. 14, 1997; Wurmet al., Genentech).

Another means to increase endogenous cyclin E2 expression in a cell viagene therapy is to insert one or more enhancer elements into the cyclinE2 promoters, where the enhancer element(s) can serve to increasetranscriptional activity of the cyclin E2 gene. The enhancer element(s)used will be selected based on the tissue in which one desires toactivate cyclin E2; enhancer elements known to confer promoteractivation in a given tissue will be selected. For example, if cyclin E2is to be "turned on" in T-cells, the lck promoter enhancer element maybe used. Here, the functional portion of the transcriptional element tobe added may be inserted into a fragment of DNA containing the cyclin E2promoter (and optionally, vector, 5' and/or 3' flanking sequence, etc aswell) using standard cloning techniques. This construct, known as a"homologous recombination construct" can then be introduced into thedesired cells either ex vivo or in vivo.

Gene therapy can be used to decrease cyclin E2 expression by modifyingthe nucleotide sequence of the endogenous cyclin E2 promoter. Suchmodification is typically accomplished via homologous recombinationmethods. For example, a DNA molecule containing all or a portion of thecyclin E2 promoter sequence can be engineered to remove and/or replacepieces of the promoter that regulate transcription. Here, the TATA boxand/or the binding site of a transcriptional activator protein of thecyclin E2 promoter may be deleted using standard molecular biologytechniques; such deletion can inhibit promoter activity therebyrepressing transcription of the corresponding cyclin E2 gene. Deletionof the TATA box or transcription activator binding site in the promotermay be accomplished by generating a DNA construct comprising all or therelevant portion of the cyclin E2 promoter (from the same or a relatedspecies as the cyclin E2 gene to be regulated) in which one or more ofthe TATA box and/or transcriptional activator binding site nucleotidesare mutated via substitution, deletion and/or insertion of one or morenucleotides such that the TATA box and/or activator binding site hasdecreased activity or is rendered completely inactive. This construct,which also will typically contain at least about 500 bases of DNA thatcorresponds to the native (endogenous) 5' and 3' flanking regions of thepromoter segment that has been modified, may be introduced into theappropriate cells (either ex vivo or in vivo) either directly or via aviral vector as described above. Typically, integration of the constructinto the genomic DNA of the cells will be via homologous recombination,where the 5' and 3' flanking DNA sequences in the promoter construct canserve to help integrate the modified promoter region via hybridizationto the endogenous chromosomal DNA.

Other gene therapy methods may also be employed where it is desirable toinhibit cyclin E2 activity. For example, antisense DNA or RNA molecules,which have a sequence that is complementary to at least a portion of thecyclin E2 can be introduced into the cell. Typically, the antisensemolecule will be complementary to the start site (5' end) of the cyclinE2 gene. When the antisense molecule then hybridizes to the cyclin E2mRNA, translation of the cyclin E2 mRNA is prevented.

Alternatively, gene therapy may be employed to create adominant-negative inhibitor of cyclin E2. In this situation, the DNAencoding a mutant full length or truncated polypeptide of cyclin E2 canbe prepared and introduced into the cells of a patient using eitherviral or non-viral methods as described above. The cyclin E2 mutant istypically designed to (1) compete with endogenous cyclin E2 in itsbiological role; and (2) contain one or more insertions, deletions,and/or mutations as compared to wild type cyclin E2 such that it stillbinds cdk2, but does not permit formation of an active cyclin E2/cdkcomplex (see, for example Diehl et al., Mol. Cell. Biol., 17:7362-7374[1997]). This mutant cyclin E2 protein, when over-expressed in the cellsinto which it is introduced, can compete with endogenous cyclin E2protein, resulting in the formation of mutant cyclin E2/cdk2 complexesthat are inactive.

Biology of Cyclin E2

While not intending to be bound to any one theory, it is believed that,in vivo, cyclin E2 is thought to form a complex with cyclin-dependentkinase 2 ("cdk2") to form a "holoenzyme"; this holoenzyme can then bephosphorylated at position 160 (threonine) of cdk2 by a separate enzymecomplex called "cyclin activating kinase" or "cak". Upon phosphorylationof the cdk2 portion of the cyclin E2/cdk holoenzyme complex, thiscomplex can then phosphorylate its substrates, which are retinoblastomaand histone H1 via the kinase activity of the cdk2 portion of theholoenzyme.

Assays to Screen for Inhibitors of Cyclin E2

Organic (biological or synthetic) or inorganic molecules that inhibitcyclin E2 can be identified using one or more of the screening assaysdescribed below. Such molecules could be administered either in an exvivo manner, or in an in vivo manner by local or iv injection, or byoral delivery, implantation device, or the like.

For ease of reading, the following definition is used herein fordescribing the assays:

"Test molecule(s)" refers to the molecule(s) that is under evaluation asan inhibitor of cyclin E2, either by virtue of its potential ability toblock (1) the interaction of cyclin E2 with cyclin dependent kinase("cdk2"; a molecule with which it naturally associates in the cell; seeExamples herein) or (2) the interaction of cyclin E2/cdk2 with cyclinactivating kinase "cak"; a molecule that "activates" the cyclin E2/cdk2complex by phosphorylation of cdk at position 160, but only when cdk2 iscomplexed with cyclin E2.

A. In Vitro Assays using Purified Proteins

One type of in vitro assay for evaluating the efficacy of a cyclin E2inhibitor test molecule requires purified cyclin E2 and cdk. Cyclin E2polypeptide and cdk polypeptide can be recombinantly produced usingmethods described above. The gene encoding cdk is known (Ninomiya-Tsujiet al., Proc. Natl. Acad. Sci. USA, 88:9006-9010 [1991]; Tsai et al.,Nature, 353:174-177 [1991]). Recombinant cyclin E2 and cdk2 polypeptidescan be full length molecules, or biologically active variants orderivatives thereof. Host cells for recombinant production of eachpolypeptide include, without limitation, bacteria such as E. coli,yeast, insect cells (using, for example, the baculovirus system),mammalian cells, or other eukaryotic cells. The two polypeptides may beco-produced in the same host cell, where the host cell is co-transfectedwith DNA encoding each polypeptide, or in separate host cells of thesame or different species.

Each polypeptide can be purified from the host cell (or culture mediumif it is secreted); typically, this will be accomplished by expressingeach polypeptide with a "tag" sequence such as hemaglutinin ("HA"), His(polyhistidine such as hexahistidine), myc or FLAG, and purifying thetag-polypeptide via affinity chromatography using, for example, a nickelcolumn for polyhistidine, or a mono- or polyclonal antibody for myc orFLAG.

As mentioned above, the cdk portion of the cyclin E2/cdk2 holoenzymemust be phosphorylated at amino acid 160 in order for the holoenzyme tohave kinase activity; phosphorylation of cdk amino acid 160 can onlyoccur when cdk is associated with cyclin E2, thereby forming theholoenzyme. The cyclin E2/cdk complex can form spontaneously if the twopolypeptides are co-expressed in eukaryotic or prokaryotic host cells.Further, when co-expressed in a eukaryotic host cell, the host cell'smachinery can phosphorylate cdk amino acid 160, provided that the hostcell has endogenous cyclin activating kinase ("cak"), which isresponsible for such phosphorylation. The phosphorylated holoenzymecomplex (the "active holoenzyme complex") can then be purified,typically using affinity chromatography.

One preferred method for producing the active holoenzyme complex is thebaculovirus system such as the pFastBac DUAL® (Gibco/BRL LifeTechnologies, Grand Island, N.Y.). In this system, both the cdk andcyclin E2 genes can be expressed on the same vector, and the vector alsocontains a polyHis tag for ease of purification.

If the two polypeptides are produced in separate host cells of the sameor different species, they can each be purified (preferably via affinitychromatography) and then combined in solution form the holoenzymecomplex. ATP and cak (cyclin dependent activating kinase) can then beadded to the holoenzyme complex; amino acid Threonine 160 of cdk canthen be phosphorylated via cak, thereby generating an active holoenzymecomplex. Once the active holoenzyme complex has been prepared andisolated, various in vitro assays for cyclin E2 inhibitors can beconducted.

In one such assay, the active holoenzyme complex is placed in solution.Gamma-labeled ATP (such as 32P-ATP), holoenzyme substrate (such ashistone H1 or retinoblastoma peptide), and the test molecule(s) can beadded to the solution either simultaneously or successively. After aperiod of incubation, the substrate can be isolated and assayed for theamount of label it contains.

In one preferred assay, termed the "scintillation proximity assay", or"SPA" (Cook, Drug Discovery Today, 1:287-294 [1996]), biotinylatedsubstrate (histone H1 or retinoblastoma peptide, for example) isattached to non-porous beads coated with streptavidin and filled withscintillation fluid. The beads can be incubated with active holoenzymecomplex, gamma-labeled ATP, and the test molecule(s) using microtiterplates (such as 96 well plates or 384 well plates). When a radiolabeledphosphate group is transferred to the substrate via the kinase activityof the active holoenzyme complex, the photon released by the radioactivephosphate group is recorded by a scintillation counter. Those wells thatcontain test molecules which are effective in inhibiting cyclin E2 suchthat the kinase activity of the holoenzyme complex is disrupted willhave fewer radioactive counts detected than control wells.

Other in vitro assays can also be conducted to evaluate test molecules.In one such assay, the substrate can be attached to wells of amicrotiter plate, and active holoenzyme complex, gamma-labeled ATP (orother suitable detection agent), and the test molecule(s) can be addedsequentially or simultaneously. After a short incubation (on the orderof seconds to minutes), the solution can be removed from each well andthe plates can be washed and then measured for the amount of labeledgamma phosphate added to the substrate by the activity of the holoenzymecomplex.

Other variations on these assays will be apparent to the ordinaryskilled artisan. For example, the substrate can be attached to beads asan alternative to attaching it to the bottom of each well; the beads canthen be removed from solution after incubation with the test molecule,labeled ATP, and active holoenzyme complex, and measuring the amount oflabel incorporated in to the substrate.

Typically, in each type of assay, the test molecule will be evaluatedover a range of concentrations, and a series of suitable controls can beused for accuracy in evaluating the results. In some cases, it may beuseful to evaluate two or more test molecules together to assay for thepossibility of "synergistic" effects.

B. In Vitro Assays using Cultured Cells

Cultured eukaryotic cell lines that are actively dividing may be used toassay for the efficacy of a test molecule in inhibiting cyclin E2.Preferred cell lines are those such as mammalian Saos-2 cells (AmericanType Culture Collection, 10801 University Blvd., Manassas, Va., USA;accession number HTB-85) and human embryonic kidney 293T cells (AmericanType Culture Collection, accession number CRL-1573), which expressdetectable levels of cyclin E2, cdk, and cak. However, actively dividingcells into which the genes encoding cyclin E2 and/or cdk are transfectedand expressed at a detectable level, but which express endogenous cak(or into which cak genes are also transfected and expressed) are alsosuitable for use in these assays.

Cell cultures can be exposed to the test molecule(s) for a predeterminedamount of time (usually up to about 24 hours). The cells can be washedto remove remaining test molecule, and then measured for their abilityto divide. Actively dividing cells can be identified in several ways.One preferred way is to incubate them for a short time in a compoundsuch as bromo-deoxyuridine (BRDU), a nucleotide analog that isincorporated into DNA as it replicates; BRDU can be detected using afluorescent-labeled antibody. Alternatively, or additionally, dividingcells can be detected using the Alamar Blue Assay® (Biosource Intl.,Camarillo, Calif.) Effective test molecules will be those which decreasethe amount of cell division as compared to control assays.

While not intending to be bound to a particular mechanism of action, itis believed that inhibition of cyclin E2 may lead to cell death, orapoptosis. Therefore, cultured cells that have been exposed to one ormore test molecules can also be evaluated for survival by use of trypanblue, or other dye or fluorescent molecule that is selectively excludedor taken up by live cells.

Other cell-based assays for detection of test molecule efficacy willreadily be apparent to the ordinary skilled artisan.

The assays can be conducted using 96 well microtiter plates or othersuitable plates that permit several assays to be conductedsimultaneously. Typically, in each type of cell culture assay, the testmolecule will be evaluated over a range of concentrations, and a seriesof "control wells" lacking either the test molecules, or the culturedcells, can be used for accuracy in evaluating the results. In somecases, it may be useful to evaluate two or more test molecules togetherto assay for the possibility of "synergistic" effects.

C. In Vivo Assays

Once test molecules have been identified, they can be evaluated forefficacy in rodent tumor models (see for example, O'Reilly et al., Cell,88:277-285 [1997]). The test molecule can be administered prior to tumoronset in the rodent; after such administration, appearance of tumors canbe monitored and compared against a control rodent not receiving thetest molecule. Alternatively, the test molecule can be administeredafter onset of the tumor, and tumor size can be monitored

The following examples are intended for illustration purposes only, andshould not be construed as limiting the scope of the invention in anyway.

EXAMPLES

1. Identification of Human Cyclin E2

An Amgen, Inc. internal EST ("expressed sequence tags") database,containing cDNAs from more than 200 different libraries, was searchedusing a peptide sequence designed to identify cyclin box-like domains.

A murine EST called BmmE7-133-G12 was obtained from this search, and wasused to search the public database Genbank which contains human DNAsequences. One sequence, accession number R84331, was obtained. Thissequence was used to design PCR primers to conduct a transcript survey.

The transcript survey was carried out by PCR using various Marathon®cDNA libraries (Clontech, Palo Alto, Calif.) with the following primers:

    GAA GAG AAT GTC AAG ACG AAG AAG CC                         (SEQ ID NO:10)

    GCT TAA ATC AGG CAA AGG TGA AGG AT                         (SEQ ID NO:11)

The conditions for the PCR were: 2.5 ul cDNA, 20 pmols of each primer,0.5 ul 50× dNTPs, 0.5 ul AmpliTaq® enzyme (Perkin Elmer), 2.5 ul of 10×PCR buffer in a final volume of 25 ul. The cycle parameters were 94 C.for 2 minutes, followed by 35 cycles of 94 C. for 30 seconds, 60 C. for30 seconds, 72 C. for 45 seconds, followed by 72 C. for 7 minutes afterthe last cycle. Fetal liver, fetal lung and thymus cDNAs were positivefor cyclin transcript.

PCR of a Fetal Liver Marathon cDNA Library® (Clontech, Palo Alto,Calif.) was used to obtain the complete coding region of the cyclin E2gene. The sense and anti-sense primers for this reaction were,respectively:

    GAA GAG AAT GTC AAG ACG AAG AAG CC                         (SEQ ID NO:10)

    CCA TCC TAA TAC GAC TCA CTA TAG GGC                        (SEQ ID NO:12)

The conditions for the PCR were: 2.5 ul cDNA, 5 pmols of each primer,0.5 ul 50× dNTPs, 0.5 ul KlenTaq® polymerase (Clontech, Palo Alto,Calif.), 2.5 ul of 10× PCR buffer in a final volume of 25 ul. The cycleparameters were 94 C. for 1 minute followed by 35 cycles of 94 C. for 30seconds, followed by 68 C. for 3 minutes. PCR products were then run ona 1 percent agarose gel. A PCR product of approximately 2.58 kilo-basepairs (kb) was cut from the gel, and the gel slices were solubilized insodium iodide and incubated at about 48 C. for about 1 hour. The DNA wasthen extracted and purified using the GeneClean® kit (Bio 101, Vista,Calif.) following the manufacturer's protocol, and ligated into thevector pCR2.1 (Invitrogen, Carlsbad, Calif.). This vector containing theinsert was then transformed into E. coli Inv-alpha-f' cells (Invitrogen,Carlsbad, Calif.) for amplification. Plasmid was purified from the cellsusing the standard alkaline lysis method with the Qiagen Max1® kit(Qiagen, Santa Clarita, Calif.), and the insert was sequenced.Sequencing indicated that the insert contained the full length humancyclin E2 cDNA.

To confirm that the sequence obtained was correct, PCR was conductedusing a high fidelity polymerase as follows. Fetal Lung and ThymusMarathon Libraries® (Clontech) were used as template, and the primersfor this reaction were:

    GAA GAG AAT GTC AAG ACG AAG AAG CC                         (SEQ ID NO:10)

    CAG TTC TAC CCA ATC TTG GTG AAT                            (SEQ ID NO:13)

The conditions for the PCR were as follows;

2.5 ul cDNA, 5 pmols of each primer, 0.5 ul 50× dNTPs, 0.5 ul PFUpolymerase (Stratagene, La Jolla, Calif.), 2.5 ul of 10× PCR buffer to afinal volume of 25 ul. The cycle parameters were 94 C. for 2 minutesfollowed by 35 cycles of 94 C. for 30 seconds, 60 C. for 30 seconds, and72 C. for 3 minutes. The resulting approximately 1.3 kb DNA band wasresolved on an agarose gel and purified as descried above. The purifiedDNA product was subcloned into pCR2.1 as described above.

Digestion of 2 independent clones with the restriction enzyme EcoRIrevealed one fragment of approximately 1.3 kb, and one fragment ofapproximately 1.2 kb. The sequence analysis of the two fragmentsrevealed a full length cyclin E2 clone (the 1.3 kb fragment), and acyclin E2 mutant clone (the 1.2 kb fragment) with an in-frame deletionof about 135 bp in the cyclin box lacking bases 496-631 of the fulllength clone.

The sequence of full length human cyclin E2 cDNA is set forth in FIG. 1(SEQ ID NO:1), and the putative amino acid sequence, as translated fromthis cDNA, is set forth in FIG. 3 (SEQ ID NO:3). The cDNA sequence ofthe splice variant is set forth in FIG. 5 (SEQ ID NO:5), and the aminoacid sequence of this splice variant is set forth in FIG. 6 (SEQ IDNO:6).

Analysis of this gene, and comparison with genes in the databases setforth above indicated that it was indeed a novel gene, with about 49percent overall identity at both the DNA and amino acid sequence levelsto human cyclin E1, and about 70 percent identity to the cyclin boxdomain of human cyclin E1 at the amino acid level. Due to the homologywith cyclin E1, it was determined that this gene was a member of thecyclin family. Therefore, it was termed "cyclin E2".

The mutant cyclin E2 clone, which is an in-frame splice variant, lacksamino acids 166-212 (bases 496-631), which are located within the cyclinbox. As described below in detail, this isoform does not bind cdk2.

A search of this human cyclin E2 cDNA sequence with the external,publicly available databases such as Genbank revealed an EST in thedatabase that has homology to the 5' end of human cyclin E2 (Genbankaccession number R84331) However, the Genbank sequence has severalincorrect bases when compared to the same stretch of nucleotides in thepresent invention.

2. Cloning of Murine Cyclin E2

The murine EST BmmE7-133-Gl2 described above was used to design thefollowing PCR primers:

    ATT TAA GCT GGG CAT GTT CAC AGG A                          (SEQ ID NO:14)

    GTC TTC AGC TTC ACT GGA CTC ACA CTT                        (SEQ ID NO:15)

A mouse brain Marathon Library® cDNA (Clontech, Palo Alto, Calif.) wasused as the PCR template. The conditions for the PCR were as follows: 5ul cDNA, 20 pmols of each primer, 0.5 ul 50× dNTPs, 0.5 ul AmpliTaq®(Roche Molecular Systems, Inc., Branchburg, N.J.), and 5 ul of 10× PCRbuffer in a final volume of 50 ul. Reaction parameters were: 94 C. for 2minutes followed by 35 cycles of 94 C. for 20 seconds, 60 C. for 30seconds, 72 C. for 50 seconds, and a 72 C./for 7 minutes hold after thelast cycle. The PCR product was a fragment of about 723 bp, andcontained residues 308 to 1031. This fragment was cloned into pCR2.1(Invitrogen, Carlsbad, Calif.) and was transformed into E. coli cells asdescribed above. After culturing the cells to amplify the plasmid, theplasmid was purified from the cells as described above, and theapproximately 723 bp insert was digested with EcoRI which generated anapproximately 153 bp fragment (residues 879 to 1031) and anapproximately 570 bp (residues 308 to 878) fragment. The 570 bp fragmentwas gel purified using procedures described above, and was used as a DNAprobe to screen a Uni-Zap® mouse testes XR library (Stratagene, LaJolla, Calif., catalog no. 937308). The library was plated out toapproximately 1×10(6) PFUs (plaque forming units), and duplicate plaquelifts were prepared using charged nylon membranes (BioRad, Hercules,Calif.). The mouse cyclin E2 570 bp probe was radiolabeled using theRediprimer® kit (Amersham Life Science, Arlington Heights, Ill.)following the manufactuere's protocol. The specific activity of theprobe was about 1×10(9) cpm/microgram. Hybridization of the filters wasconducted overnight at about 42 C. in standard hybridization solutioncontaining 2.5×Denhardt's solution, 50 percent formamide, 0.1 percentSDS, 5×SSC, and 0.1 mg/ml salmon sperm DNA. After hybridization, thefilters were washed twice in 2×SSC at room temperature for about 15minutes, followed by three washes in 2×SSC at about 50 C. for about 30minutes, and then once in 0.2×SSC plus 0.5 percent SDS at about 42 C.for 30 minutes. The filters were exposed to film at about minus 70 C.for about 3 days using intensifying screens.

Six positives were identified from this hybridization screen. The insertof each clone was subcloned into the vector pBluescript (Stratagene, LaJolla, Calif.) using standard techniques and each insert was sequenced.

The sequence of the murine cyclin E2 cDNA is set forth in FIG. 2 (SEQ IDNO:2). The putative amino acid sequence, as translated from the cDNA, isset forth in FIG. 4 (SEQ ID NO:4). Murine cyclin E2 had about 89 percentidentity to human cyclin E2 at the DNA level, and about 92 percentidentity to human cyclin E2 at the amino acid level.

3. Human Cyclin E2 Protein Preparation

Full length human cyclin E2 polypeptide was prepared as aglutathione-s-transferase fusion protein using the PGEX® system(Pharmacia, Piscataway, N.J.) and following the manufacturer's protocol.The cyclin E2 cDNA was inserted into the vector pGEX-4T-2 at the 3' endof the DNA encoding GST. The vector was transformed into E coli strainBL-21 cells (Invitrogen, San Diego, Calif.) that were transformationcompetent by mixing plasmid DNA with the cells. After culturing thecells overnight, the cells were diluted 1:100 in media and cultured forabout 4 hours at 37 C., after which IPTG was added at a finalconcentration of 0.1 mM. After about 4 hours of culturing, proteinextract was prepared from the cells using the GST Gene Fusion System®protocol (Pharmacia Biotech, Piscataway, N.J. following themanufacturer's protocol, and this extract was then added to a slurry ofGlutathione-Agarose 4B Sepharose beads (Pharmacia Biotech, Piscataway,N.J.). The extract solution containing the beads was then centrifuged,and the beads were collected and washed. These beads were then mixedwith a slurry of agarose beads to which thrombin protease was bound(Pharmacia Biotech, Piscataway, N.J.). After mixing and incubating thetwo sets of beads for about 1 hour at room temperature, the solution wascentrifuged to pellet the beads. Samples of the supernatant were run onSDS-PAGE to assess purity. A photo of a Coomassie stained gel is shownin FIG. 9. As can be seen, only a single band of about 46 kDa waspresent; this band corresponds to the expected molecular weight forcyclin E2, indicating that the procedure resulted in generation ofpurified cyclin E2 polypeptide. To confirm that this band was indeedcyclin E2, a Western blot was prepared and probed with anti-cyclin E2antiserum (using antiserum raised against the cyclin E2 peptide of SEQID NO:17).

4. Human Cyclin E2 Antibody Production

Rabbit polyclonal antiserum raised against two cyclin E2 peptides wereprepared as follows. Peptides corresponding to the following two aminoacid sequences were prepared using standard peptide synthesisprocedures.

    GQL SPV CNG GIM TPP KST EK                                 (SEQ ID NO:16)

    AKQ QPQ PSQ TES PQE AQI IQA                                (SEQ ID NO:17)

About 5 mg of each peptide was mixed with Freund's complete adjuvant ina total volume of about 1 ml, and injected subcutaneously into rabbits.After about 4 weeks, the rabbits were again injected with the samesolution. After 2 weeks, the rabbits were bled and the serum was testedfor antibodies to the peptides by Western blot. A third injection wasadministered after the test bleed, and about two weeks later, a secondtest bleed was conducted. About two weeks later, a fourth injection wasadministered, and about two weeks after that, the final blood collectionwas conducted.

Blood obtained from the final bleed was allowed to clot by incubating itat about 4 C. for about 2 hours. The serum was then obtained bycentrifugation and collecting the supernatant. This serum was used forWestern blots and immunoprecipitations described herein.

5. Human Cyclin E2 Expression in Mammalian Cells

Human full length cyclin E2 cDNA was inserted into the plasmid pEGFP-n1(Invitrogen, San Diego, Calif.) which contains the coding region forgreen fluorescent protein ("GFP"). The cyclin E2 cDNA was inserted 3' tothe GFP DNA to generate the vector pGFP-E2.

About 5 micrograms of plasmid was then transfected into about 2×10(6)human embryonic kidney 293T cells using the standard calcium phosphatetransfection procedure.

Simultaneously, the cells were transfected with a vector, calledHA-cdk2, containing the gene encoding human cdk2 (Turner et al., Genesand Devel., 8:1434-1447 [1994]).

After transfection, the cells were incubated at about 37 C. for about 24hours, after which the cells were harvested into about 200 microlitersof a buffer termed "TG buffer" containing 1 percent Triton X-100, 10percent glycerol, 0.1 percent SDS, 0.5 percent deoxycholate, 20 mM HepespH 7.4, 100 mM NaCl, and about 1 mM each of leupeptin, aprotinin, PMSF,sodium vanadate, and sodium fluoride. Cell debris was removed bycentrifugation at about 10,000×g, after which the supernatant wascollected and the pellet discarded.

To the extract was added about 1 microliter of anti-GFP polyclonalantiserum (Invitrogen, San Diego, Calif.) and the extract was incubatedat about 4 C. for about 1 hour. After incubation, about 50 microlitersof Protein A/Protein G agarose beads (Pierce Biochemicals, Rockford,Ill.) was added, and the mixture was incubated about 30 minutes at about4 C. After this incubation, the beads were pelleted and washed fivetimes with TG buffer minus SDS and deoxycholate. After washing, thebeads were resuspended in standard SDS-PAGE sample buffer, heated atabout 95 C. for about 3 minutes, centrifuged, and the supernatant wascollected and loaded on to a 12 percent SDS-PAGE gel.

The gel was transferred to PVDF membrane (NEN Life Sciences, Burton,Mass.) using standard Western blotting procedures, and the membrane wascut into strips for probing with different antibodies including anti-GFP(Invitrogen, San Diego, Calif.), anti-HA (Beohringer-Mannheim,Indianapolis, Ind.), and anti-p27 (Santa Cruz Biologicals, Santa Cruz,Calif.).

Separately, the same procedure was followed for the human cyclin E2splice variant, and the same procedures as those described immediatelyabove for the wild type cyclin E2 transfected cells were conducted forthese cells in order to determine whether cdk binds to the splicevariant.

In one other separate set of experiments, a DNA construct containing acDNA encoding human p27 (Polyak et al., Cell, 78:59-66 [1994]) wasco-transfected to cells that were also transfected with full lengthcyclin E2 and cdk2. The procedures followed for this transfection werethe same as those set forth above.

The results of Western blot analysis are shown in FIGS. 10A-D. As shownin 10A, GFP protein fused to either full length cyclin E2 or the splicevariant "sv" cyclin E2 is immunoprecipitated using anti-GFP antiserum.IgG heavy chain is also detected in the Western blot as indicated. FIG.10B shows that GFP alone is also immunoprecipitated by the anti-GFPantiserum. FIG. 10C shows that cdk2 associates (and thusco-immunoprecipitates) with wild type cyclin E2 but not with the cyclinE2 splice variant, and therefore, the middle portion of the cyclin boxdomain, which is missing in the splice variant, is likely responsiblefor cdk2 binding to cyclin E2. FIG. 10D shows that in a three waytransfection of the cells with cdk2, cyclin E2 (full length) and p27 DNAconstructs, p27 co-immunoprecipitates with the other two proteins,indicating that p27 forms a complex with cdk2 and cyclin E2. FIG. 10Eshows that a complex containing full length cyclin E2 and cdk2 canphosphorylate histone H1, and that the presence of p27 decreases theamount of phosphorylation. In addition, the cyclin E2 splicevariant-cdk2 complex cannot phosphorylate histone H1. The experimentsfor this kinase assay were conducted as described immediately below.

6. Biological Activity of Cyclin E2

Biological activity of cyclin E2/cdk2 was evaluated using a kinaseassay. Extracts were prepared by treating about 2×10 (6) Saos-2 cells(American Type Culture Collection, 10801 University Blvd., Manassas,Va., USA) with about 200 microliters of TG buffer (see above), andcentrifuging the extract to pellet cellular debris. About 3 microlitersof antibody (either anti cyclin E2 raised against the peptide of SEQ IDNO:17, anti-GFP, or preimmune serum "PI") was added to about 500micrograms of cell extract protein, and the mixture was incubated about1 hour at about 4 C. About 50 microliters of Protein A/G Sepharose beadswas then added, and this mixture was incubated about 30 minutes at about4 C. The beads were then washed about 4 times with TG buffer (about 500microliters per wash) after which kinase buffer containing 50 mM Tris pH7.5, 10 mM MgCl2, 1 mM DTT, 25 mM ATP, and 40 micro-CI of gamma-32P-ATPwas added to the extract. Various potential kinase substrates were thenadded, including histone HI, GST-retinoblastoma (pRb; Santa CruzBiotechnology, Santa Cruz, Calif.), or GST-p53 (Santa CruzBiotechnology). The mixture was then incubated at about 37 C. for about30 minutes, after which standard 2× SDS-PAGE sample buffer was added.The samples were heated at about 95 C. for about 3 minutes, run onSDS-PAGE, and exposed to film for 2 hours. The results can be seen inFIG. 15, Panel B. As is apparent, the cyclin E2-cdk2 complex canphosphorylate GST-Rb and Histone Hi, but not GST-p53.

7. Tissue Specific Expression of Cyclin E2

Northern blot analysis was used to identify those tissues in whichcyclin E2 is expressed. A Northern blot containing about 2 micrograms ofpoly A+RNA from various human tissues was purchased (Clontech, PaloAlto, Calif.) and was probed with either an approximately 320 bp humancyclin E2 cDNA fragment spanning a region 5' to the cyclin box, a 310base pair cyclin E1 fragment spanning amino acids 272-377 of humancyclin E1, or a human GADPH (glyceraldehydephospho-dehydrogenase) probeof about 550 bp encoding a portion of the human GADPH gene. The probeswere labeled using the RediPrimer® kit (Amersham, Arlington Heights,Ill.), and the final activity of the labeled probes was about 1×10(9)cpm per microgram. The blots were prehybridized about 2 hours, andhybridized overnight at about 42 C. in a hybridization solutioncontaining 2×Denhardt's, 0.5 percent SDS, 50 percent formamide, 0.1 mgper microgram of salmon sperm DNA, and 5×SSC. The specific activity ofthe probe in this solution was about 2×10(6) cpm. After hybridization,the blots were washed three times in 2×SSC containing 0.1 percent SDS atroom temperature, twice in 2×SSC containing 0.1 percent SDS at 50 C.,and once in 0.1×SSC containing 0.5 percent SDS at 50 C. Films wereexposed at about minus 70 C. for two days. The resulting autorads werescanned into a computer, and the data was converted to a bar graph toquantitate the relative amounts of RNA detected by each probe.

The results are shown in FIG. 12, and the human tissues analyzed areindicated on the x-axis. AS can be seen, testes tissue had the highestlevel of both cyclin E2 and cyclin E1 RNA. Cyclin E1 RNA was present inperipheral blood lymphocytes ("PBL"), but cyclin E2 RNA was not. CyclinE2 RNA was present in spleen and thymus, but cyclin E1 RNA was not.

To assess cyclin E2 RNA levels in tumor tissues, several breast cancercell lines, together with normal immortalized breast tissue cell lines,were evaluated. The cell lines are listed in FIG. 13. All cell lines areavailable from the American Type Culture Collection.

The NMEC, 184A1 and MCF10 cells were cultured in modified DME/F12(Gibco/BRL, Grand Island, N.Y.) medium supplemented with 10 mM Hepes, 2mM glutamine, 0.1 mM non-essential amino acids, 0.5 mM ethanolamine, 5micrograms/ml transferrin, 1 mg/ml bovine serum albumin, 5.0 ng/mlsodium selenite, 20 ng/ml triiodothyronine, 10 ng/ml EGF, 5micrograms/ml insulin, and 0.5 micrograms/ml hydrocortisone. Bovinepituitary extract (30 micrograms/ml) was also added to the medium forthe NMEC cell lines. The ER+ and ER- (estrogen receptor positive andnegative) breast cancer cell lines were cultured in alpha or Richterimproved minimal essential medium (Gibco/BRL, Grand Island, N.Y.)supplemented with 10 mM Hepes, 2 mM glutamine, 0.1 mM non-essentialamino acids, 10 percent fetal bovine serum, and 1 microgram/ml insulin.All cells were routinely screened for mycoplasma contamination, and weremaintained at 37 C. in an atmosphere of about 6.5 percent carbondioxide.

Total RNA was prepared from each cell line by lysing cell monolayers inguanidinium isothiocyanate and centrifuging over a cesium chloridecushion as described by Gudas et al. (Proc. Natl. Acad. Sci USA, 3585:4705-4709 [1988]). About 20 micrograms of RNA was electrophoresed ondenaturing formaldehyde gels, transferred to Magna NT membranes (MicronSeparations Inc., Westboro, Mass.), and cross linked with UV radiation.The probes for Northern blot analysis included the full length cyclin E1cDNA, and an approximately 330 bp fragment of cyclin E2 (as describedabove). The probes were labeled with 32P-dCTP to a specific activity ofabout 1×10(9) cpm/microgram DNA using a random-primed labeling kit(Boehringer-Mannheim, Indianapolis, Ind.). The blot was hybridized in asolution containing 50 percent formamide, 0.2 percent SDS, 6×SSPE,2×Denhardt's and 100 ng/ml salmon sperm DNA. Hybridization was conductedat about 41 C. for about 24 hours, after which the blot was washed oncein 2×SSC containing 0.5 percent SDS at room temperature; once in 0.5×SSCcontaining 0.5 percent SDS at room temperature; once in 0.2×SSCcontaining 0.5 percent SDS at room temperature; and three times in0.5×SSC containing 0.5 percent SDS at about 59 C.

The results of Northern analysis are shown in FIG. 13. AS can be seen,cyclin E2 RNA is present in some ER+ and some ER- cell lines, but ishardly detectable in normal cells. Cyclin E1 RNA is present in fewerbreast cancer cell lines as compared to cyclin E2.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 18                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 1215                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Human                                                          - - <400> SEQUENCE: 1                                                         - - atgtcaagac gaagtagccg tttacaagct aagcagcagc cccagcccag cc -            #agacggaa     60                                                                 - - tccccccaag aagcccagat aatccaggcc aagaagagga aaactaccca gg -            #atgtcaaa    120                                                                 - - aaaagaagag aggaggtcac caagaaacat cagtatgaaa ttaggaattg tt -            #ggccacct    180                                                                 - - gtattatctg gggggatcag tccttgcatt atcattgaaa cacctcacaa ag -            #aaatagga    240                                                                 - - acaagtgatt tctccagatt tacaaattac agatttaaaa atctttttat ta -            #atccttca    300                                                                 - - cctttgcctg atttaagctg gggatgttca aaagaagtct ggctaaacat gt -            #taaaaaag    360                                                                 - - gagagcagat atgttcatga caaacatttt gaagttctgc attctgactt gg -            #aaccacag    420                                                                 - - atgaggtcca tacttctaga ctggctttta gaggtatgtg aagtatacac ac -            #ttcatagg    480                                                                 - - gaaacatttt atcttgcaca agactttttt gatagattta tgttgacaca aa -            #aggatata    540                                                                 - - aataaaaata tgcttcaact cattggaatt acctcattat tcattgcttc ca -            #aacttgag    600                                                                 - - gaaatctatg ctcctaaact ccaagagttt gcttacgtca ctgatggtgc tt -            #gcagtgaa    660                                                                 - - gaggatatct taaggatgga actcattata ttaaaggctt taaaatggga ac -            #tttgtcct    720                                                                 - - gtaacaatca tctcctggct aaatctcttt ctccaagttg atgctcttaa ag -            #atgctcct    780                                                                 - - aaagttcttc tacctcagta ttctcaggaa acattcattc aaatagctca gc -            #ttttagat    840                                                                 - - ctgtgtattc tagccattga ttcattagag ttccagtaca gaatactgac tg -            #ctgctgcc    900                                                                 - - ttgtgccatt ttacctccat tgaagtggtt aagaaagcct caggtttgga gt -            #gggacagt    960                                                                 - - atttcagaat gtgtagattg gatggtacct tttgtcaatg tagtaaaaag ta -            #ctagtcca   1020                                                                 - - gtgaagctga agacttttaa gaagattcct atggaagaca gacataatat cc -            #agacacat   1080                                                                 - - acaaactatt tggctatgct ggaggaagta aattacataa acaccttcag aa -            #aaggggga   1140                                                                 - - cagttgtcac cagtgtgcaa tggaggcatt atgacaccac cgaagagcac tg -            #aaaaacca   1200                                                                 - - ccaggaaaac actaa              - #                  - #                      - #  1215                                                                  - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 1212                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Mouse                                                          - - <400> SEQUENCE: 2                                                         - - atgtcaagac gcagccgttt acaagctaag caacatgccc agcccaacca gc -             #cagactct     60                                                                 - - ccgcaagaaa cccagataat tcaggccaag aagagaaaaa cagcacagga tg -            #tcaaaaaa    120                                                                 - - agaaaagagg agatcaccaa gaagcatcag tatgagatta ggaattgttg gc -            #cacctgta    180                                                                 - - ctgtctggag gaatcagccc ttgcattatc attgaaacac cccataaaga aa -            #taggaaca    240                                                                 - - agtgacttct ctagatttac aaattacaga tttaaaaatc tttttattaa tc -            #cctcacct    300                                                                 - - ctgccagatt taagctgggc atgttcacag gaggtttggc aaaacatgtt ac -            #aaaaggaa    360                                                                 - - aacagatacg tgcatgacaa acattttcaa gttctgcatt ctgacctgga ac -            #cacagatg    420                                                                 - - aggtcaatac ttttagactg gcttttagag gtttgtgaag tatacactct tc -            #atagggag    480                                                                 - - acattttacc ttgcccaaga cttttttgac agatttatgt tgacacaaaa gg -            #atgtaaat    540                                                                 - - aaaaatatgc ttcaactcat tgggattacc tcattgttca ttgcttccaa ac -            #ttgaggaa    600                                                                 - - atctacgctc ctaaactcca agagtttgct tacgtcactg atggtgcttg ca -            #gtgaagta    660                                                                 - - gatatcttaa agatggaact caatatatta aaggctttaa aatgggaact tt -            #gtccagta    720                                                                 - - acagtcatct cctggttgaa tctttttctt caagttgatg ctgttaaaga tg -            #ttcctaag    780                                                                 - - gttcttctac ctcaatattc tcaggagacg ttcatccaga tagctcagct tt -            #tagatctg    840                                                                 - - tgcattctag ccatcgactc tttagaattt caatacagaa ttctggctgc tg -            #ccgcctta    900                                                                 - - tgtcatttta cctccattga agtggttaag aaagcttcag gtttggaatg gg -            #atgacatc    960                                                                 - - tcggaatgtg tagactggat ggtgcctttt gttagtgttg taaaaagtgt ga -            #gtccagtg   1020                                                                 - - aagctgaaga cttttaagaa gatacccatg gaagatagac acaatatcca ga -            #cacacaca   1080                                                                 - - aattatttgg ctttgctgaa tgaagtaaac tatgtgaaca tctacagaaa ag -            #gagggcag   1140                                                                 - - ctgtcaccag tgtgtaatgg aggcattatg acaccaccaa agagtactga aa -            #aaccacca   1200                                                                 - - ggaaaacact ga              - #                  - #                      - #     1212                                                                  - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 404                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Human                                                          - - <400> SEQUENCE: 3                                                         - - Met Ser Arg Arg Ser Ser Arg Leu Gln Ala Ly - #s Gln Gln Pro Gln Pro        1               5 - #                 10 - #                 15              - - Ser Gln Thr Glu Ser Pro Gln Glu Ala Gln Il - #e Ile Gln Ala Lys Lys                   20     - #             25     - #             30                  - - Arg Lys Thr Thr Gln Asp Val Lys Lys Arg Ar - #g Glu Glu Val Thr Lys               35         - #         40         - #         45                      - - Lys His Gln Tyr Glu Ile Arg Asn Cys Trp Pr - #o Pro Val Leu Ser Gly           50             - #     55             - #     60                          - - Gly Ile Ser Pro Cys Ile Ile Ile Glu Thr Pr - #o His Lys Glu Ile Gly       65                 - # 70                 - # 75                 - # 80       - - Thr Ser Asp Phe Ser Arg Phe Thr Asn Tyr Ar - #g Phe Lys Asn Leu Phe                       85 - #                 90 - #                 95              - - Ile Asn Pro Ser Pro Leu Pro Asp Leu Ser Tr - #p Gly Cys Ser Lys Glu                  100      - #           105      - #           110                  - - Val Trp Leu Asn Met Leu Lys Lys Glu Ser Ar - #g Tyr Val His Asp Lys              115          - #       120          - #       125                      - - His Phe Glu Val Leu His Ser Asp Leu Glu Pr - #o Gln Met Arg Ser Ile          130              - #   135              - #   140                          - - Leu Leu Asp Trp Leu Leu Glu Val Cys Glu Va - #l Tyr Thr Leu His Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Thr Phe Tyr Leu Ala Gln Asp Phe Phe As - #p Arg Phe Met Leu        Thr                                                                                             165  - #               170  - #               175             - - Gln Lys Asp Ile Asn Lys Asn Met Leu Gln Le - #u Ile Gly Ile Thr Ser                  180      - #           185      - #           190                  - - Leu Phe Ile Ala Ser Lys Leu Glu Glu Ile Ty - #r Ala Pro Lys Leu Gln              195          - #       200          - #       205                      - - Glu Phe Ala Tyr Val Thr Asp Gly Ala Cys Se - #r Glu Glu Asp Ile Leu          210              - #   215              - #   220                          - - Arg Met Glu Leu Ile Ile Leu Lys Ala Leu Ly - #s Trp Glu Leu Cys Pro      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Val Thr Ile Ile Ser Trp Leu Asn Leu Phe Le - #u Gln Val Asp Ala        Leu                                                                                             245  - #               250  - #               255             - - Lys Asp Ala Pro Lys Val Leu Leu Pro Gln Ty - #r Ser Gln Glu Thr Phe                  260      - #           265      - #           270                  - - Ile Gln Ile Ala Gln Leu Leu Asp Leu Cys Il - #e Leu Ala Ile Asp Ser              275          - #       280          - #       285                      - - Leu Glu Phe Gln Tyr Arg Ile Leu Thr Ala Al - #a Ala Leu Cys His Phe          290              - #   295              - #   300                          - - Thr Ser Ile Glu Val Val Lys Lys Ala Ser Gl - #y Leu Glu Trp Asp Ser      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ile Ser Glu Cys Val Asp Trp Met Val Pro Ph - #e Val Asn Val Val        Lys                                                                                             325  - #               330  - #               335             - - Ser Thr Ser Pro Val Lys Leu Lys Thr Phe Ly - #s Lys Ile Pro Met Glu                  340      - #           345      - #           350                  - - Asp Arg His Asn Ile Gln Thr His Thr Asn Ty - #r Leu Ala Met Leu Glu              355          - #       360          - #       365                      - - Glu Val Asn Tyr Ile Asn Thr Phe Arg Lys Gl - #y Gly Gln Leu Ser Pro          370              - #   375              - #   380                          - - Val Cys Asn Gly Gly Ile Met Thr Pro Pro Ly - #s Ser Thr Glu Lys Pro      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Pro Gly Lys His                                                           - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 403                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Mouse                                                          - - <400> SEQUENCE: 4                                                         - - Met Ser Arg Arg Ser Arg Leu Gln Ala Lys Gl - #n His Ala Gln Pro        Asn                                                                               1               5 - #                 10 - #                 15             - - Gln Pro Asp Ser Pro Gln Glu Thr Gln Ile Il - #e Gln Ala Lys Lys Arg                   20     - #             25     - #             30                  - - Lys Thr Ala Gln Asp Val Lys Lys Arg Lys Gl - #u Glu Ile Thr Lys Lys               35         - #         40         - #         45                      - - His Gln Tyr Glu Ile Arg Asn Cys Trp Pro Pr - #o Val Leu Ser Gly Gly           50             - #     55             - #     60                          - - Ile Ser Pro Cys Ile Ile Ile Glu Thr Pro Hi - #s Lys Glu Ile Gly Thr       65                 - # 70                 - # 75                 - # 80       - - Ser Asp Phe Ser Arg Phe Thr Asn Tyr Arg Ph - #e Lys Asn Leu Phe Ile                       85 - #                 90 - #                 95              - - Asn Pro Ser Pro Leu Pro Asp Leu Ser Trp Al - #a Cys Ser Gln Glu Val                  100      - #           105      - #           110                  - - Trp Gln Asn Met Leu Gln Lys Glu Asn Arg Ty - #r Val His Asp Lys His              115          - #       120          - #       125                      - - Phe Gln Val Leu His Ser Asp Leu Glu Pro Gl - #n Met Arg Ser Ile Leu          130              - #   135              - #   140                          - - Leu Asp Trp Leu Leu Glu Val Cys Glu Val Ty - #r Thr Leu His Arg Glu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Thr Phe Tyr Leu Ala Gln Asp Phe Phe Asp Ar - #g Phe Met Leu Thr        Gln                                                                                             165  - #               170  - #               175             - - Lys Asp Val Asn Lys Asn Met Leu Gln Leu Il - #e Gly Ile Thr Ser Leu                  180      - #           185      - #           190                  - - Phe Ile Ala Ser Lys Leu Glu Glu Ile Tyr Al - #a Pro Lys Leu Gln Glu              195          - #       200          - #       205                      - - Phe Ala Tyr Val Thr Asp Gly Ala Cys Ser Gl - #u Val Asp Ile Leu Lys          210              - #   215              - #   220                          - - Met Glu Leu Asn Ile Leu Lys Ala Leu Lys Tr - #p Glu Leu Cys Pro Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Thr Val Ile Ser Trp Leu Asn Leu Phe Leu Gl - #n Val Asp Ala Val        Lys                                                                                             245  - #               250  - #               255             - - Asp Val Pro Lys Val Leu Leu Pro Gln Tyr Se - #r Gln Glu Thr Phe Ile                  260      - #           265      - #           270                  - - Gln Ile Ala Gln Leu Leu Asp Leu Cys Ile Le - #u Ala Ile Asp Ser Leu              275          - #       280          - #       285                      - - Glu Phe Gln Tyr Arg Ile Leu Ala Ala Ala Al - #a Leu Cys His Phe Thr          290              - #   295              - #   300                          - - Ser Ile Glu Val Val Lys Lys Ala Ser Gly Le - #u Glu Trp Asp Asp Ile      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ser Glu Cys Val Asp Trp Met Val Pro Phe Va - #l Ser Val Val Lys        Ser                                                                                             325  - #               330  - #               335             - - Val Ser Pro Val Lys Leu Lys Thr Phe Lys Ly - #s Ile Pro Met Glu Asp                  340      - #           345      - #           350                  - - Arg His Asn Ile Gln Thr His Thr Asn Tyr Le - #u Ala Leu Leu Asn Glu              355          - #       360          - #       365                      - - Val Asn Tyr Val Asn Ile Tyr Arg Lys Gly Gl - #y Gln Leu Ser Pro Val          370              - #   375              - #   380                          - - Cys Asn Gly Gly Ile Met Thr Pro Pro Lys Se - #r Thr Glu Lys Pro Pro      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Gly Lys His                                                               - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 1080                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Human                                                          - - <400> SEQUENCE: 5                                                         - - atgtcaagac gaagtagccg tttacaagct aagcagcagc cccagcccag cc -            #agacggaa     60                                                                 - - tccccccaag aagcccagat aatccaggcc aagaagagga aaactaccca gg -            #atgtcaaa    120                                                                 - - aaaagaagag aggaggtcac caagaaacat cagtatgaaa ttaggaattg tt -            #ggccacct    180                                                                 - - gtattatctg gggggatcag tccttgcatt atcattgaaa cacctcacaa ag -            #aaatagga    240                                                                 - - acaagtgatt tctccagatt tacaaattac agatttaaaa atctttttat ta -            #atccttca    300                                                                 - - cctttgcctg atttaagctg gggatgttca aaagaagtct ggctaaacat gt -            #taaaaaag    360                                                                 - - gagagcagat atgttcatga caaacatttt gaagttctgc attctgactt gg -            #aaccacag    420                                                                 - - atgaggtcca tacttctaga ctggctttta gaggtatgtg aagtatacac ac -            #ttcatagg    480                                                                 - - gaaacatttt atcttgctta cgtcactgat ggtgcttgca gtgaagagga ta -            #tcttaagg    540                                                                 - - atggaactca ttatattaaa ggctttaaaa tgggaacttt gtcctgtaac aa -            #tcatctcc    600                                                                 - - tggctaaatc tctttctcca agttgatgct cttaaagatg ctcctaaagt tc -            #ttctacct    660                                                                 - - cagtattctc aggaaacatt cattcaaata gctcagcttt tagatctgtg ta -            #ttctagcc    720                                                                 - - attgattcat tagagttcca gtacagaata ctgactgctg ctgccttgtg cc -            #attttacc    780                                                                 - - tccattgaag tggttaagaa agcctcaggt ttggagtggg acagtatttc ag -            #aatgtgta    840                                                                 - - gattggatgg taccttttgt caatgtagta aaaagtacta gtccagtgaa gc -            #tgaagact    900                                                                 - - tttaagaaga ttcctatgga agacagacat aatatccaga cacatacaaa ct -            #atttggct    960                                                                 - - atgctggagg aagtaaatta cataaacacc ttcagaaaag ggggacagtt gt -            #caccagtg   1020                                                                 - - tgcaatggag gcattatgac accaccgaag agcactgaaa aaccaccagg aa -            #aacactaa   1080                                                                 - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 359                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Human                                                          - - <400> SEQUENCE: 6                                                         - - Met Ser Arg Arg Ser Ser Arg Leu Gln Ala Ly - #s Gln Gln Pro Gln        Pro                                                                               1               5 - #                 10 - #                 15             - - Ser Gln Thr Glu Ser Pro Gln Glu Ala Gln Il - #e Ile Gln Ala Lys Lys                   20     - #             25     - #             30                  - - Arg Lys Thr Thr Gln Asp Val Lys Lys Arg Ar - #g Glu Glu Val Thr Lys               35         - #         40         - #         45                      - - Lys His Gln Tyr Glu Ile Arg Asn Cys Trp Pr - #o Pro Val Leu Ser Gly           50             - #     55             - #     60                          - - Gly Ile Ser Pro Cys Ile Ile Ile Glu Thr Pr - #o His Lys Glu Ile Gly       65                 - # 70                 - # 75                 - # 80       - - Thr Ser Asp Phe Ser Arg Phe Thr Asn Tyr Ar - #g Phe Lys Asn Leu Phe                       85 - #                 90 - #                 95              - - Ile Asn Pro Ser Pro Leu Pro Asp Leu Ser Tr - #p Gly Cys Ser Lys Glu                  100      - #           105      - #           110                  - - Val Trp Leu Asn Met Leu Lys Lys Glu Ser Ar - #g Tyr Val His Asp Lys              115          - #       120          - #       125                      - - His Phe Glu Val Leu His Ser Asp Leu Glu Pr - #o Gln Met Arg Ser Ile          130              - #   135              - #   140                          - - Leu Leu Asp Trp Leu Leu Glu Val Cys Glu Va - #l Tyr Thr Leu His Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Thr Phe Tyr Leu Ala Tyr Val Thr Asp Gl - #y Ala Cys Ser Glu        Glu                                                                                             165  - #               170  - #               175             - - Asp Ile Leu Arg Met Glu Leu Ile Ile Leu Ly - #s Ala Leu Lys Trp Glu                  180      - #           185      - #           190                  - - Leu Cys Pro Val Thr Ile Ile Ser Trp Leu As - #n Leu Phe Leu Gln Val              195          - #       200          - #       205                      - - Asp Ala Leu Lys Asp Ala Pro Lys Val Leu Le - #u Pro Gln Tyr Ser Gln          210              - #   215              - #   220                          - - Glu Thr Phe Ile Gln Ile Ala Gln Leu Leu As - #p Leu Cys Ile Leu Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ile Asp Ser Leu Glu Phe Gln Tyr Arg Ile Le - #u Thr Ala Ala Ala        Leu                                                                                             245  - #               250  - #               255             - - Cys His Phe Thr Ser Ile Glu Val Val Lys Ly - #s Ala Ser Gly Leu Glu                  260      - #           265      - #           270                  - - Trp Asp Ser Ile Ser Glu Cys Val Asp Trp Me - #t Val Pro Phe Val Asn              275          - #       280          - #       285                      - - Val Val Lys Ser Thr Ser Pro Val Lys Leu Ly - #s Thr Phe Lys Lys Ile          290              - #   295              - #   300                          - - Pro Met Glu Asp Arg His Asn Ile Gln Thr Hi - #s Thr Asn Tyr Leu Ala      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Met Leu Glu Glu Val Asn Tyr Ile Asn Thr Ph - #e Arg Lys Gly Gly        Gln                                                                                             325  - #               330  - #               335             - - Leu Ser Pro Val Cys Asn Gly Gly Ile Met Th - #r Pro Pro Lys Ser Thr                  340      - #           345      - #           350                  - - Glu Lys Pro Pro Gly Lys His                                                      355                                                                    - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 1214                                                            <212> TYPE: RNA                                                               <213> ORGANISM: Human                                                          - - <400> SEQUENCE: 7                                                         - - augucccguc guuccucccg ucugcaggcu aaacagcagc cgcagccguc cc -             #agaccgaa     60                                                                 - - uccccgcagg aagcucagau cauccaggcu aaaaaacgua aaaccaccca gg -            #acguuaaa    120                                                                 - - aaacgucgug aagaaguuac caaaaaacac caguacgaaa uccguaacug cu -            #ggccgccg    180                                                                 - - guucuguccg gugguaucuc cccgugcauc aucaucgaaa ccccgcacaa ag -            #aaaucggu    240                                                                 - - accuccgacu ucucccguuu caccaacuac cguuucaaaa accuguucau ca -            #acccgucc    300                                                                 - - ccgcugccgg accuguccug ggguugcucc aaagaaguuu ggcugaacau gc -            #ugaaaaaa    360                                                                 - - gaaucccguu acguucacga caaacacuuc gaaguucugc acuccgaccu gg -            #aaccgcag    420                                                                 - - augcguucca uccugcugga cuggcugcug gaaguuugcg aaguuuacac cc -            #ugcaccgu    480                                                                 - - gaaaccuucu accuggcuca ggacuucuuc gaccguuuca ugcugaccca ga -            #aagacauc    540                                                                 - - aacaaaaaca ugcugcagcu gaucgguauc accucccugu ucaucgcuuc ca -            #aacuggaa    600                                                                 - - gaaaucuacg cuccgaaacu gcaggaauuc gcuuacguua ccgacggugc uu -            #gcuccgaa    660                                                                 - - gaagacaucc ugcguaugga acugaucauc cugaaagcuc ugaaauggga ac -            #ugugcccg    720                                                                 - - guuaccauca ucuccuggcu gaaccuguuc cugcagguug acgcucugaa ag -            #acgcuccg    780                                                                 - - aaaguucugc ugccgcagua cucccaggaa accuucaucc agaucgcuca gc -            #ugcuggac    840                                                                 - - cugugcaucc uggcuaucga cucccuggaa uuccaguacc guauccugac cg -            #cugcugcu    900                                                                 - - cugugccacu ucaccuccau cgaaguuguu aaaaaagcuu ccggucugga au -            #gggacucc    960                                                                 - - aucuccgaau gcguugacug gaugguuccg uucguuaacg uuguuaaauc ca -            #ccuccccg   1020                                                                 - - guuaaacuga aaaccuucaa aaaaaucccg auggaagacc gucacaacau cc -            #agacccac   1080                                                                 - - accaacuacc uggcuaugcu ggaagaaguu aacuacauca acaccuuccg ua -            #aagguggu   1140                                                                 - - cagcuguccc cgguuugcaa cggugguauc augaccccgc cgaaauccac cg -            #aaaaaccg   1200                                                                 - - ccggguaaac acug              - #                  - #                      - #   1214                                                                  - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 1215                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Human                                                          - - <400> SEQUENCE: 8                                                         - - atgwsnmgnm gnwsnwsnmg nytncargcn aarcarcarc cncarccnws nc -             #aracngar     60                                                                 - - wsnccncarg argcncarat hathcargcn aaraarmgna aracnacnca rg -            #aygtnaar    120                                                                 - - aarmgnmgng argargtnac naaraarcay cartaygara thmgnaaytg yt -            #ggccnccn    180                                                                 - - gtnytnwsng gnggnathws nccntgyath athathgara cnccncayaa rg -            #arathggn    240                                                                 - - acnwsngayt tywsnmgntt yacnaaytay mgnttyaara ayytnttyat ha -            #ayccnwsn    300                                                                 - - ccnytnccng ayytnwsntg gggntgywsn aargargtnt ggytnaayat gy -            #tnaaraar    360                                                                 - - garwsnmgnt aygtncayga yaarcaytty gargtnytnc aywsngayyt ng -            #arccncar    420                                                                 - - atgmgnwsna thytnytnga ytggytnytn gargtntgyg argtntayac ny -            #tncaymgn    480                                                                 - - garacnttyt ayytngcnca rgayttytty gaymgnttya tgytnacnca ra -            #argayath    540                                                                 - - aayaaraaya tgytncaryt nathggnath acnwsnytnt tyathgcnws na -            #arytngar    600                                                                 - - garathtayg cnccnaaryt ncargartty gcntaygtna cngayggngc nt -            #gywsngar    660                                                                 - - gargayathy tnmgnatgga rytnathath ytnaargcny tnaartggga ry -            #tntgyccn    720                                                                 - - gtnacnatha thwsntggyt naayytntty ytncargtng aygcnytnaa rg -            #aygcnccn    780                                                                 - - aargtnytny tnccncarta ywsncargar acnttyathc arathgcnca ry -            #tnytngay    840                                                                 - - ytntgyathy tngcnathga ywsnytngar ttycartaym gnathytnac ng -            #cngcngcn    900                                                                 - - ytntgycayt tyacnwsnat hgargtngtn aaraargcnw snggnytnga rt -            #gggaywsn    960                                                                 - - athwsngart gygtngaytg gatggtnccn ttygtnaayg tngtnaarws na -            #cnwsnccn   1020                                                                 - - gtnaarytna aracnttyaa raarathccn atggargaym gncayaayat hc -            #aracncay   1080                                                                 - - acnaaytayy tngcnatgyt ngargargtn aaytayatha ayacnttymg na -            #arggnggn   1140                                                                 - - carytnwsnc cngtntgyaa yggnggnath atgacnccnc cnaarwsnac ng -            #araarccn   1200                                                                 - - ccnggnaarc aytrr              - #                  - #                      - #  1215                                                                  - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: synthetic construct                                            - - <400> SEQUENCE: 9                                                         - - gaagagaatg tcaagacgaa gaagcc          - #                  - #                  26                                                                      - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: synthetic construct                                            - - <400> SEQUENCE: 10                                                        - - gcttaaatca ggcaaaggtg aaggat          - #                  - #                  26                                                                      - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 27                                                              <212> TYPE: DNA                                                               <213> ORGANISM: synthetic construct                                            - - <400> SEQUENCE: 11                                                        - - ccatcctaat acgactcact atagggc          - #                  - #                 27                                                                      - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: synthetic construct                                            - - <400> SEQUENCE: 12                                                        - - cagttctacc caatcttggt gaat          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: synthetic construct                                            - - <400> SEQUENCE: 13                                                        - - atttaagctg ggcatgttca cagga          - #                  - #                   25                                                                      - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 27                                                              <212> TYPE: DNA                                                               <213> ORGANISM: synthetic construct                                            - - <400> SEQUENCE: 14                                                        - - gtcttcagct tcactggact cacactt          - #                  - #                 27                                                                      - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 20                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Human                                                          - - <400> SEQUENCE: 15                                                        - - Gly Gln Leu Ser Pro Val Cys Asn Gly Gly Il - #e Met Thr Pro Pro Lys        1               5 - #                 10 - #                 15              - - Ser Thr Glu Lys                                                                       20                                                                - -  - - <210> SEQ ID NO 16                                                  <211> LENGTH: 21                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Human                                                          - - <400> SEQUENCE: 16                                                        - - Ala Lys Gln Gln Pro Gln Pro Ser Gln Thr Gl - #u Ser Pro Gln Glu Ala        1               5 - #                 10 - #                 15              - - Gln Ile Ile Gln Ala                                                                   20                                                                - -  - - <210> SEQ ID NO 17                                                  <211> LENGTH: 1212                                                            <212> TYPE: RNA                                                               <213> ORGANISM: Mouse                                                          - - <400> SEQUENCE: 17                                                        - - augucccguc guucccgucu gcaggcuaaa cagcacgcuc agccgaacca gc -             #cggacucc     60                                                                 - - ccgcaggaaa cccagaucau ccaggcuaaa aaacguaaaa ccgcucagga cg -            #uuaaaaaa    120                                                                 - - cguaaagaag aaaucaccaa aaaacaccag uacgaaaucc guaacugcug gc -            #cgccgguu    180                                                                 - - cuguccggug guaucucccc gugcaucauc aucgaaaccc cgcacaaaga aa -            #ucgguacc    240                                                                 - - uccgacuucu cccguuucac caacuaccgu uucaaaaacc uguucaucaa cc -            #cguccccg    300                                                                 - - cugccggacc uguccugggc uugcucccag gaaguuuggc agaacaugcu gc -            #agaaagaa    360                                                                 - - aaccguuacg uucacgacaa acacuuccag guucugcacu ccgaccugga ac -            #cgcagaug    420                                                                 - - cguuccaucc ugcuggacug gcugcuggaa guuugcgaag uuuacacccu gc -            #accgugaa    480                                                                 - - accuucuacc uggcucagga cuucuucgac cguuucaugc ugacccagaa ag -            #acguuaac    540                                                                 - - aaaaacaugc ugcagcugau cgguaucacc ucccuguuca ucgcuuccaa ac -            #uggaagaa    600                                                                 - - aucuacgcuc cgaaacugca ggaauucgcu uacguuaccg acggugcuug cu -            #ccgaaguu    660                                                                 - - gacauccuga aaauggaacu gaacauccug aaagcucuga aaugggaacu gu -            #gcccgguu    720                                                                 - - accguuaucu ccuggcugaa ccuguuccug cagguugacg cuguuaaaga cg -            #uuccgaaa    780                                                                 - - guucugcugc cgcaguacuc ccaggaaacc uucauccaga ucgcucagcu gc -            #uggaccug    840                                                                 - - ugcauccugg cuaucgacuc ccuggaauuc caguaccgua uccuggcugc ug -            #cugcucug    900                                                                 - - ugccacuuca ccuccaucga aguuguuaaa aaagcuuccg gucuggaaug gg -            #acgacauc    960                                                                 - - uccgaaugcg uugacuggau gguuccguuc guuuccguug uuaaauccgu uu -            #ccccgguu   1020                                                                 - - aaacugaaaa ccuucaaaaa aaucccgaug gaagaccguc acaacaucca ga -            #cccacacc   1080                                                                 - - aacuaccugg cucugcugaa cgaaguuaac uacguuaaca ucuaccguaa ag -            #guggucag   1140                                                                 - - cuguccccgg uuugcaacgg ugguaucaug accccgccga aauccaccga aa -            #aaccgccg   1200                                                                 - - gguaaacacu ga              - #                  - #                      - #     1212                                                                  - -  - - <210> SEQ ID NO 18                                                  <211> LENGTH: 1212                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Mouse                                                          - - <400> SEQUENCE: 18                                                        - - atgwsnmgnm gnwsnmgnyt ncargcnaar carcaygcnc arccnaayca rc -             #cngaywsn     60                                                                 - - ccncargara cncarathat hcargcnaar aarmgnaara cngcncarga yg -            #tnaaraar    120                                                                 - - mgnaargarg arathacnaa raarcaycar taygarathm gnaaytgytg gc -            #cnccngtn    180                                                                 - - ytnwsnggng gnathwsncc ntgyathath athgaracnc cncayaarga ra -            #thggnacn    240                                                                 - - wsngayttyw snmgnttyac naaytaymgn ttyaaraayy tnttyathaa yc -            #cnwsnccn    300                                                                 - - ytnccngayy tnwsntgggc ntgywsncar gargtntggc araayatgyt nc -            #araargar    360                                                                 - - aaymgntayg tncaygayaa rcayttycar gtnytncayw sngayytnga rc -            #cncaratg    420                                                                 - - mgnwsnathy tnytngaytg gytnytngar gtntgygarg tntayacnyt nc -            #aymgngar    480                                                                 - - acnttytayy tngcncarga yttyttygay mgnttyatgy tnacncaraa rg -            #aygtnaay    540                                                                 - - aaraayatgy tncarytnat hggnathacn wsnytnttya thgcnwsnaa ry -            #tngargar    600                                                                 - - athtaygcnc cnaarytnca rgarttygcn taygtnacng ayggngcntg yw -            #sngargtn    660                                                                 - - gayathytna aratggaryt naayathytn aargcnytna artgggaryt nt -            #gyccngtn    720                                                                 - - acngtnathw sntggytnaa yytnttyytn cargtngayg cngtnaarga yg -            #tnccnaar    780                                                                 - - gtnytnytnc cncartayws ncargaracn ttyathcara thgcncaryt ny -            #tngayytn    840                                                                 - - tgyathytng cnathgayws nytngartty cartaymgna thytngcngc ng -            #cngcnytn    900                                                                 - - tgycayttya cnwsnathga rgtngtnaar aargcnwsng gnytngartg gg -            #aygayath    960                                                                 - - wsngartgyg tngaytggat ggtnccntty gtnwsngtng tnaarwsngt nw -            #snccngtn   1020                                                                 - - aarytnaara cnttyaaraa rathccnatg gargaymgnc ayaayathca ra -            #cncayacn   1080                                                                 - - aaytayytng cnytnytnaa ygargtnaay taygtnaaya thtaymgnaa rg -            #gnggncar   1140                                                                 - - ytnwsnccng tntgyaaygg nggnathatg acnccnccna arwsnacnga ra -            #arccnccn   1200                                                                 - - ggnaarcayt rr              - #                  - #                      - #     1212                                                                __________________________________________________________________________

We claim:
 1. An isolated biologically active cyclin E2 nucleic acidmolecule encoding a polypeptide selected from the group consistingof:(a) the nucleic acid molecule comprising SEQ ID NO:2; (b) the nucleicacid molecule comprising SEQ ID NO:5; (c) the nucleic acid moleculecomprising SEQ ID NO:7; (d) the nucleic acid molecule comprising SEQ IDNO:8; (e) a nucleic acid molecule encoding the polypeptide of SEQ IDNO:3; (f) a nucleic acid molecule encoding the polypeptide of SEQ IDNO:4; (g) a nucleic acid molecule encoding the polypeptide of SEQ IDNO:6; (h) a nucleic acid molecule that encodes a polypeptide or abiologically active fragment thereof, wherein said polypeptide or saidbiologically active fragment is at least 70 percent identical to thepolypeptide of SEQ ID NO:3 as calculated using the computer algorithmBLAST2 with its default parameters together with the scoring matrixBLOSUM 62; (i) a nucleic acid molecule that encodes a polypeptide or abiologically active fragment thereof that is at least 70 percentidentical to the polypeptide of SEQ ID NO:4 as calculated using thecomputer algorithm BLAST2 with its default parameters together with thescoring matrix BLOSUM 62; (j) a nucleic acid molecule that encodes apolypeptide or a biologically active fragment thereof that is at least70 percent identical to the polypeptide of SEQ ID NO:6 as calculatedusing the computer algorithm BLAST2 with its default parameters togetherwith the scoring matrix BLOSUM 62; and (k) a nucleic acid molecule thathybridizes to any of (a)-(i) above under washing conditions of 0.2×SSCand 0.1 percent SDS at a temperature of 50-65° C.
 2. An isolated nucleicacid molecule that is the complement of the full nucleic acid moleculeof claim
 1. 3. An isolated nucleic acid molecule comprising SEQ ID NO:2,SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8.
 4. An isolated nucleic acidmolecule encoding the polypeptide of SEQ ID NO:3, SEQ ID NO:4, or SEQ IDNO:6.
 5. A vector comprising the nucleic acid molecule of claim
 1. 6. Avector comprising the nucleic acid molecule of claim
 2. 7. A vectorcomprising the nucleic acid molecule of claim
 3. 8. A vector comprisingthe nucleic acid molecule of claim
 4. 9. A host cell comprising thevector of claim
 5. 10. A host cell comprising the vector of claim
 6. 11.A host cell comprising the vector of claim
 7. 12. A host cell comprisingthe vector of claim
 8. 13. A process for producing a cyclin E2polypeptide comprising the steps of:(a) expressing a polypeptide encodedby the nucleic acid of claim 1 in a suitable host; and (b) isolating thepolypeptide.
 14. The process of claim 13 wherein the polypeptide is SEQID NO:3, SEQ ID NO:4, or SEQ ID NO:6.